Therapeutic compositions for viral-associated disease states and methods of making and using same

ABSTRACT

A method comprising obtaining a bodily fluid from a subject; contacting the bodily fluid with an adsorbent material comprising a synthetic carbon particle (SCP) to produce a first filtrate having a level of disease mediators (y); contacting the first filtrate with an adsorbent material comprising the SCP and an anion exchange resin where the ratio of SCP to anion exchange resin is from about 0.1:100 to 100:0.1 to produce a second filtrate; contacting the second filtrate with an adsorbent material comprising the SCP and a cation exchange resin where the ratio of SCP to cation exchange resin is from about 1:100 to produce a third filtrate; and administering the third filtrate to the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of and claims priority toU.S. patent application Ser. No. 14/642,538 filed on Mar. 9, 2015, whichclaims priority to U.S. Provisional Application No. 61/981,061, filed onApr. 17, 2014 and entitled “Plasma Detoxification.” and U.S. ProvisionalApplication No. 62/055,392, filed on Sep. 25, 2014 and entitled“Therapeutic Compositions for Viral Associated Disease States andMethods of Making and Using Same,” each of which is incorporated byreference herein in its entirety.

TECHNICAL FIELD

Generally disclosed herein are compositions, systems, and methods forthe treatment of subjects experiencing a viral-associated disease state.More specifically disclosed herein are methodologies for the treatmentof subjects suffering from immune suppression as the result of a viralinfection.

BACKGROUND

Suppression of the immune system is a common symptom observed inindividuals infected with viruses such as the Ebola virus, Lassa virus,and Marburg virus. Ebola virus, Lassa Virus, and Marburg virus belong toa taxonomically diverse set of single-stranded ribonucleic acid (ssRNA)viruses from four diverse viral families Arenaviridae, Bunyaviridae,Filoviridae, and Flaviviridae. These viruses cause an acute systemicfebrile syndrome called viral hemorrhagic fever (VHF). Other examples ofviruses that cause immune system suppression include the Hantaviruseswhich are single-stranded, enveloped, negative sense RNA viruses in theBunyaviridae family; MERS-coronavirus (MERS-CoV) which is abetacornavirus derived from bat; Influenza A virus subtype H5N1, alsoknown as A(H5N1) or simply H5N1, which is a subtype of the influenza Avirus that can cause illness in humans and many other animal species;and Influenza A (H1N1) virus which is an othomyxovirus and a subtype ofinfluenza A virus that was the most common cause of human influenza(flu) in 2009.

A need exits for additional methods of effectively treating subjectssuffering from infection with these viruses. For example, a recentsevere outbreak of VHF on the African continent has captured globalattention. The World Health Organization (WHO) cites the first cases ofthe largest and most complex Ebola outbreak to date were noted in March2014. The gravity of the situation surrounding the recent Ebola outbreakreflects an urgent need for effective, inexpensive, and robustcompositions and methodologies for the treatment of subjects sufferingfrom viruses such as Ebola, Hanta, MERS, and influenza.

SUMMARY

Disclosed herein is a method comprising obtaining a bodily fluid from asubject having a level of disease mediators (y); contacting the bodilyfluid with an adsorbent material comprising a synthetic carbon particle(SCP) to produce a first filtrate; contacting the first filtrate with anadsorbent material comprising the SCP and an anion exchange resin wherethe weight ratio of SCP to anion exchange resin is from about 0.1:100 to100:0.1 produce a second filtrate; contacting the second filtrate withan adsorbent material comprising the SCP and a cation exchange resinwhere the weight ratio of SCP to cation exchange resin is from about0.1:100 to 100:0.1 produce a third filtrate; and administering the thirdfiltrate to the subject.

Also disclosed herein is an extracorporeal system comprising at leastthree adsorbent materials, an access disconnection detector, and acomputer system.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present disclosure and theadvantages thereof, reference is now made to the following briefdescription, taken in connection with the accompanying drawings anddetailed description, wherein like reference numerals represent likeparts.

FIGS. 1 and 2 depict embodiments of an apparatus disclosed herein.

FIG. 3A is a plot of the amount of TNFα in a sample of fresh frozenplasma as a function of time following contact with the indicatedadsorbent.

FIG. 3B is a plot of the amount of IL-1β in a sample of fresh frozenplasma as a function of time following contact with the indicatedadsorbent.

FIG. 3C is a plot of the amount of IL-6 in a sample of fresh frozenplasma as a function of time following contact with the indicatedadsorbent.

FIG. 3D is a plot of the amount of IL-8 in a sample of fresh frozenplasma as a function of time following contact with the indicatedadsorbent.

FIG. 3E is a plot of the amount of IL-10 in a sample of fresh frozenplasma as a function of time following contact with the indicatedadsorbent.

FIG. 3F is a plot of the amount of MCP-1 in a sample of fresh frozenplasma as a function of time following contact with the indicatedadsorbent.

FIG. 3G is a plot of the amount of IFNγ in a sample of fresh frozenplasma as a function of time following contact with the indicatedadsorbent.

FIG. 3H is a plot of the amount of C-reactive protein in a sample offresh frozen plasma as a function of time following contact with theindicated adsorbent.

FIG. 4A is a plot of the amount of nitrite in a sample of fresh frozenplasma as a function of time following contact with the indicatedadsorbent.

FIG. 4B is a plot of the amount of nitrate in a sample of fresh frozenplasma as a function of time following contact with the indicatedadsorbent.

FIG. 4C is a plot of the amount of endotoxin in a sample of fresh frozenplasma as a function of time following contact with the indicatedadsorbent.

FIG. 4D is a plot of the amount of hydrogen peroxide in a sample offresh frozen plasma as a function of time following contact with theindicated adsorbent.

FIG. 5A is a plot of the amount of TNFα, IL-1β, and IL-6 in a sample ofwhole blood as a function of time following contact with the indicatedadsorbent.

FIG. 5B is a plot of the amount of IL-8 in a sample of fresh frozenplasma as a function of time following contact with the indicatedadsorbent.

FIG. 5C is a plot of the amount of IL-10, MCP-1 and MCP1α in a sample offresh frozen plasma as a function of time following contact with theindicated adsorbent.

FIG. 5D is a plot of the amount of TGF-β and IFNγ in a sample of freshfrozen plasma as a function of time following contact with the indicatedadsorbent.

FIG. 5E is a plot of the amount of nitric oxide and hydrogen peroxide ina sample of fresh frozen plasma as a function of time following contactwith the indicated adsorbent.

FIG. 5F is a plot of the amount of endotoxin in a sample of fresh frozenplasma as a function of time following contact with the indicatedadsorbent.

FIGS. 6A and 6B are plots of the amount of albumin in a sample of wholeblood as a function of time following exposure to an adsorbent that hadbeen primed with the indicated amount of dextran.

DETAILED DESCRIPTION

Disclosed herein are methodologies and compositions useful for thetreatment of subjects suffering from a viral-associated disease statethat can result in immunosuppression, such as for example infection withthe Ebola virus, Marburg virus, and Hanta virus. Hereinafter, suchviruses which cause an infection that can result in immunosuppressiveevents in the infected subject are collectively termedviruses-associated with immunosuppressive events (VISE). Also disclosedherein are apparatuses useful in the treatment of subjects sufferingfrom a VISE. In an embodiment, the compositions disclosed hereincomprise adsorbent materials that are utilized in conjunction with oneor more apparatuses of the type disclosed herein in the treatment ofsubject suffering from a VISE. Herein the term adsorbent material isused for simplicity and it is to be understood the term “adsorbent” doesnot necessarily refer to the mechanism of action of the material.

The term “subject,” as used herein, comprises any and all organisms andincludes the term “patient.” A subject to be treated according to themethods described herein may be one who has been diagnosed by a medicalpractitioner as being infected with a VISE. Diagnosis may be performedby any suitable means. A subject in whom the development of an infectionis being prevented may or may not have received such a diagnosis. Oneskilled in the art will understand that a subject to be treatedaccording to the present disclosure may have been subjected to standardtests to diagnose an infection with a VISE or may have been identified,without examination, as one at high risk for infection with a VISE dueto the presence of one or more risk factors (e.g., proximity to orcontact with the bodily fluids of a subject known to be infected withVISE etc.).

Herein “treating” refers to utilizing the disclosed methodologies andcompositions for prophylactic and/or therapeutic purposes. Prophylactictreatment may be administered, for example, to a subject who is not yetill, but who is susceptible to, or otherwise at risk of a particulardisorder, e.g., VISE. Therapeutic treatment may be administered, forexample, to a subject already suffering from a disorder in order toimprove or stabilize the subject's condition (e.g., a patient alreadysuffering from a VISE). Thus, in the claims and embodiments describedherein, treating refers to a subject undergoing, either for therapeuticor prophylactic purposes, the methodologies disclosed herein.

In some instances, as compared with an equivalent untreated control,treatment may ameliorate a disorder or a symptom thereof. As usedherein, amelioration of the disorder or symptoms thereof by undergoingthe methodologies disclosed herein refers to any lessening, whetherlasting or transient, which can be attributed to or associated withundergoing the methodologies disclosed herein. In some instances,treating can result in the inhibition of viral infection, the treatmentof the infection, and/or the amelioration of symptoms of the infection.Confirmation of treatment can be assessed by detecting an improvement inor the absence of symptoms, or by the inability to detect the presenceof the infectious agent and/or disease mediators in the treated subject.

In an embodiment, a method of the present disclosure comprises (i)contacting the bodily fluid of a subject with an apparatus for removalof one or more components present in the bodily fluid to produce adecontaminated bodily fluid; and returning at least a portion of thedecontaminated bodily fluid to a subject. As used herein the term“bodily fluid,” includes inter alia plasma, whole blood, andcerebrospinal fluid. In an embodiment, at least a portion of the bodilyfluid is removed from the test subject. In an embodiment, the bodilyfluid comprises whole blood or plasma.

In an alternative embodiment, a method of the present disclosurecomprises contacting at least a portion of a subject's blood with anapparatus for removal of one or more materials present in the blood toproduce decontaminated blood; and returning at least a portion of thedecontaminated blood to the subject.

In an alternative embodiment, a method of the present disclosurecomprises contacting at least a portion of the blood of a subjectsuffering from a VISE with an apparatus of the type disclosed herein.The method may further comprise recovering at least a portion of thesubject's blood to obtain a decontaminated blood. The method may furthercomprise administering at least a portion of the decontaminated blood tothe subject.

In an alternative embodiment, a method of the present disclosurecomprises identifying a subject suffering from or at risk for thedevelopment of a VISE. The method may further comprise performingextracorporeal cleansing of at least a portion of the subject's bloodutilizing the apparatuses and compositions disclosed herein to generatea decontaminated blood. The method may further comprise administering atleast a portion of the decontaminated blood to the subject.

In an embodiment, a method comprises obtaining a blood sample from asubject diagnosed with and/or at risk for development of a VISE. Themethodologies disclosed herein may be utilized in the treatment ofsubjects infected with viruses from viral families such as Arenaviridae,Bunyaviridae, Filoviridae, Flaviviridae, Coronavirinae, Bunyaviridae,and Orthomyxoviridae. For example, the subject may be infected with aVISE such as the Hantavirus, MERS-coronavirus (MERS-CoV), Influenza Avirus subtype H5N1, Influenza A (H1N1) virus, Ebola Virus, Marburgvirus, Lassa virus, or combinations thereof

Hereinafter for simplicity, the disclosure may refer specifically to thetreatment of Ebola infected subjects, although it is to be understoodthe subject matter disclosed herein may be utilized in the treatment ofVISEs from other viral sources such as those disclosed herein. Diagnosisof and/or assessment of the risk for development of a VISE may be madeby a healthcare professional using any suitable methodology. As known tothe ordinarily skilled artisan, the clinical features of a VISE varyaccording to the source of the virus.

In an embodiment, a blood sample is obtained from a subject who is influid communication with an extracorporeal apparatus. The subject may beinfected with a VISE or may be suspected of being infected with a VISE.An embodiment of an apparatus suitable for use in the present disclosureis depicted in FIG. 1. In an embodiment, the apparatus 300 comprises aninlet 305 in fluid communication with a pump 360 which regulates accessand fluid communication with conduit 315. The apparatus 300 may beconnected to a subject through establishing a means of blood flow fromthe subject to inlet 305. An arterial access of the subject 355 may beused to establish a means of blood flow from the subject to the inlet305. As a safety measure, apparatus 300 in one embodiment includes aplurality of electrodes (not shown), such as two to four electrodes,which provide an access disconnection sensor, which is integrated halfin the arterial line 305 and half in the venous line 392 to detectaccess disconnection of the subject from the apparatus 300. Analternative embodiment for detection of accidental needle disconnectionsis the use of a conductive blanket underneath the subject's access. Insuch embodiments, the presence of blood changes the conductivity of theblanket and sets off an alarm and stops the pumps.

With reference to FIG. 1, a methodology of the type disclosed hereincomprises establishing fluid communication between a subject's bloodflow as accessed through a vein (e.g., jugular, subclavian or femoralveins) of the subject 355 and the inlet 305 of the apparatus 300.Alternatively the subject's blood flow may be accessed via one or morechronic vascular accesses. For example, a chronic vascular access mayhave been created by a surgical procedure: such as (i) nativearteriovenous fistulas (native AVFs), (ii) arteriovenous shunts usinggraft material (AV graft), and (iii) tunnelled double-lumen catheters.The pump 360 regulates the flow of the subject's blood to the remainderof the apparatus 300 through conduit 315. Conduit 315 may be a pipe orflow line comprised of material suitable for use in the methodologiesdisclosed herein. In an embodiment, the subject's blood is allowed toflow through conduit 315 until it reaches valve 380 which when in the onposition allows the blood flow to enter column A 310 in a particularflow direction 390. Blood may be pumped through column A 310 and exitthe column thorough an outlet regulated by a valve 385. Blood exitingfrom column A 310 through the outlet regulated by valve 385 may enterconduit 395 where it is pumped to inlet port 340 whose access isregulated by valve 345. When valve 345 is in the on position, the bloodmay be pumped from inlet port 340 to column B 350 where it moves in flowdirection G 342 through column B 350 to outlet port 348 which isregulated by valve 352. When valve 352 is in the on position the bloodmay flow from column B 350 into conduit 315. In an embodiment, thesubject's blood is allowed to flow through conduit 315 until it reachesinlet port 360 which is regulated by valve 362 which when in the onposition allows the blood flow to enter column C 370 in a particularflow direction H 375. The blood may exit column C 370 via outlet port378 which is regulated by valve 376 which when in the on position allowsthe blood to flow into conduit 395 and back to the vein of the subject392.

In an embodiment, the rate of flow of a bodily fluid (e.g., blood)through apparatus 300 may be regulated to provide some user and/orprocess goal. For example, the rate of blood flow through apparatus 300may range from about 1 mL/min to about 300 mL/min, alternatively fromabout 25 mL/min to about 300 mL/min, alternatively from about 25 mL/minto about 150 mL/min, or alternatively from about 150 mL/min to about 300mL/min. In an embodiment, treatment of a subject suffering from a VISEmay require the subject be in fluid communication with apparatus 300 fora period of time ranging from about 1 hour to about 24 hours,alternatively from about 1 hour to about 12 hours, alternatively fromabout 1 hour to about 6 hours, alternatively from about 1 hour to about4 hours, or alternatively less than about 4 hours. In an embodiment, thesubject is in fluid communication with the apparatus 300 for a timeperiod sufficient to allow from about 0.5 to about 10× the total bloodvolume of the subject to circulate through the apparatus 300.Alternatively, from about 1 to about 10× the total blood volume of thesubject is allowed to circulate through the apparatus. In yet anotherembodiment, the blood volume circulated through the apparatus (e.g.,apparatus 300) may range from about 5 liters to about 72 liters,alternatively form about 10 liters to about 60 liters, or alternativelyfrom about 36 liters to about 54 liters and may occur in a time periodranging from about 1 hour to about 6 hours or alternatively from about 3hours to about 4 hours. In some embodiments, the subject suffering fromthe VISE (e.g., Ebola) may undergo treatments where they are placed influid communication with the apparatus a plurality of times as deemedsufficient to address their particular disease state.

It is to be understood that FIG. 1 presents an embodiment of anapparatus suitable for use in the present disclosure. Additional routinemodifications to the apparatus are contemplated by the presentdisclosure. For example, the apparatus may contain more or less than the3 columns depicted in FIG. 1 or the columns may be disposed in positionsother than perpendicular to conduits 315 and 395. In an embodiment, theapparatus 300 may be associated with a computer system.

FIG. 2 illustrates a computer system 780 suitable for implementing oneor more embodiments disclosed herein. The computer system 780 includes aprocessor 782 (which may be referred to as a central processor unit orCPU) that is in communication with memory devices including secondarystorage 784, read only memory (ROM) 786, random access memory (RAM) 788,input/output (I/O) devices 790, and network connectivity devices 792.The processor 782 may be implemented as one or more CPU chips.

It is understood that by programming and/or loading executableinstructions onto the computer system 780, at least one of the CPU 782,the RAM 788, and the ROM 786 are changed, transforming the computersystem 780 in part into a particular machine or apparatus having thenovel functionality taught by the present disclosure. It is fundamentalto the electrical engineering and software engineering arts thatfunctionality that can be implemented by loading executable softwareinto a computer can be converted to a hardware implementation bywell-known design rules. Decisions between implementing a concept insoftware versus hardware typically hinge on considerations of stabilityof the design and numbers of units to be produced rather than any issuesinvolved in translating from the software domain to the hardware domain.Generally, a design that is still subject to frequent change may bepreferred to be implemented in software, because re-spinning a hardwareimplementation is more expensive than re-spinning a software design.Generally, a design that is stable that will be produced in large volumemay be preferred to be implemented in hardware, for example in anapplication specific integrated circuit (ASIC), because for largeproduction runs the hardware implementation may be less expensive thanthe software implementation. Often a design may be developed and testedin a software form and later transformed, by well-known design rules, toan equivalent hardware implementation in an application specificintegrated circuit that hardwires the instructions of the software. Inthe same manner as a machine controlled by a new ASIC is a particularmachine or apparatus, likewise a computer that has been programmedand/or loaded with executable instructions may be viewed as a particularmachine or apparatus.

The secondary storage 784 is typically comprised of one or more diskdrives or tape drives and is used for non-volatile storage of data andas an over-flow data storage device if RAM 788 is not large enough tohold all working data. Secondary storage 784 may be used to storeprograms which are loaded into RAM 788 when such programs are selectedfor execution. The ROM 786 is used to store instructions and perhapsdata which are read during program execution. ROM 786 is a non-volatilememory device which typically has a small memory capacity relative tothe larger memory capacity of secondary storage 784. The RAM 788 is usedto store volatile data and perhaps to store instructions. Access to bothROM 786 and RAM 788 is typically faster than to secondary storage 784.The secondary storage 784, the RAM 788, and/or the ROM 786 may bereferred to in some contexts as computer readable storage media and/ornon-transitory computer readable media.

I/O devices 790 may include printers, video monitors, liquid crystaldisplays (LCDs), touch screen displays, keyboards, keypads, switches,dials, mice, track balls, voice recognizers, card readers, paper tapereaders, or other well-known input devices.

The network connectivity devices 792 may take the form of modems, modembanks, Ethernet cards, universal serial bus (USB) interface cards,serial interfaces, token ring cards, fiber distributed data interface(FDDI) cards, wireless local area network (WLAN) cards, radiotransceiver cards such as code division multiple access (CDMA), globalsystem for mobile communications (GSM), long-term evolution (LTE),worldwide interoperability for microwave access (WiMAX), and/or otherair interface protocol radio transceiver cards, and other well-knownnetwork devices. These network connectivity devices 792 may enable theprocessor 782 to communicate with an Internet or one or more intranets.With such a network connection, it is contemplated that the processor782 might receive information from the network, or might outputinformation to the network in the course of performing theabove-described method steps. Such information, which is oftenrepresented as a sequence of instructions to be executed using processor782, may be received from and outputted to the network, for example, inthe form of a computer data signal embodied in a carrier wave.

Such information, which may include data or instructions to be executedusing processor 782 for example, may be received from and outputted tothe network, for example, in the form of a computer data baseband signalor signal embodied in a carrier wave. The baseband signal or signalembodied in the carrier wave generated by the network connectivitydevices 792 may propagate in or on the surface of electrical conductors,in coaxial cables, in waveguides, in an optical conduit, for example anoptical fiber, or in the air or free space. The information contained inthe baseband signal or signal embedded in the carrier wave may beordered according to different sequences, as may be desirable for eitherprocessing or generating the information or transmitting or receivingthe information. The baseband signal or signal embedded in the carrierwave, or other types of signals currently used or hereafter developed,may be generated according to several methods well known to one skilledin the art. The baseband signal and/or signal embedded in the carrierwave may be referred to in some contexts as a transitory signal.

The processor 782 executes instructions, codes, computer programs,scripts which it accesses from hard disk, floppy disk, optical disk(these various disk based systems may all be considered secondarystorage 784), ROM 786, RAM 788, or the network connectivity devices 792.While only one processor 782 is shown, multiple processors may bepresent. Thus, while instructions may be discussed as executed by aprocessor, the instructions may be executed simultaneously, serially, orotherwise executed by one or multiple processors. Instructions, codes,computer programs, scripts, and/or data that may be accessed from thesecondary storage 784, for example, hard drives, floppy disks, opticaldisks, and/or other device, the ROM 786, and/or the RAM 788 may bereferred to in some contexts as non-transitory instructions and/ornon-transitory information.

In an embodiment, the computer system 780 may comprise two or morecomputers in communication with each other that collaborate to perform atask. For example, but not by way of limitation, an application may bepartitioned in such a way as to permit concurrent and/or parallelprocessing of the instructions of the application. Alternatively, thedata processed by the application may be partitioned in such a way as topermit concurrent and/or parallel processing of different portions of adata set by the two or more computers. In an embodiment, virtualizationsoftware may be employed by the computer system 780 to provide thefunctionality of a number of servers that is not directly bound to thenumber of computers in the computer system 780. For example,virtualization software may provide twenty virtual servers on fourphysical computers. In an embodiment, the functionality disclosed abovemay be provided by executing the application and/or applications in acloud computing environment. Cloud computing may comprise providingcomputing services via a network connection using dynamically scalablecomputing resources. Cloud computing may be supported, at least in part,by virtualization software. A cloud computing environment may beestablished by an enterprise and/or may be hired on an as-needed basisfrom a third party provider. Some cloud computing environments maycomprise cloud computing resources owned and operated by the enterpriseas well as cloud computing resources hired and/or leased from a thirdparty provider.

In an embodiment, some or all of the functionality disclosed above maybe provided as a computer program product. The computer program productmay comprise one or more computer readable storage medium havingcomputer usable program code embodied therein to implement thefunctionality disclosed above. The computer program product may comprisedata structures, executable instructions, and other computer usableprogram code. The computer program product may be embodied in removablecomputer storage media and/or non-removable computer storage media. Theremovable computer readable storage medium may comprise, withoutlimitation, a paper tape, a magnetic tape, magnetic disk, an opticaldisk, a solid state memory chip, for example analog magnetic tape,compact disk read only memory (CD-ROM) disks, floppy disks, jump drives,digital cards, multimedia cards, and others. The computer programproduct may be suitable for loading, by the computer system 780, atleast portions of the contents of the computer program product to thesecondary storage 784, to the ROM 786, to the RAM 788, and/or to othernon-volatile memory and volatile memory of the computer system 780. Theprocessor 782 may process the executable instructions and/or datastructures in part by directly accessing the computer program product,for example by reading from a CD-ROM disk inserted into a disk driveperipheral of the computer system 780. Alternatively, the processor 782may process the executable instructions and/or data structures byremotely accessing the computer program product, for example bydownloading the executable instructions and/or data structures from aremote server through the network connectivity devices 792. The computerprogram product may comprise instructions that promote the loadingand/or copying of data, data structures, files, and/or executableinstructions to the secondary storage 784, to the ROM 786, to the RAM788, and/or to other non-volatile memory and volatile memory of thecomputer system 780.

In some contexts, a baseband signal and/or a signal embodied in acarrier wave may be referred to as a transitory signal. In somecontexts, the secondary storage 784, the ROM 786, and the RAM 788 may bereferred to as a non-transitory computer readable medium or a computerreadable storage media. A dynamic RAM embodiment of the RAM 788,likewise, may be referred to as a non-transitory computer readablemedium in that while the dynamic RAM receives electrical power and isoperated in accordance with its design, for example during a period oftime during which the computer 780 is turned on and operational, thedynamic RAM stores information that is written to it. Similarly, theprocessor 782 may comprise an internal RAM, an internal ROM, a cachememory, and/or other internal non-transitory storage blocks, sections,or components that may be referred to in some contexts as non-transitorycomputer readable media or computer readable storage media.

In an embodiment, adsorbent materials are disposed within or containedby the columns of apparatus 300. Adsorbent materials suitable for use inthe present disclosure include chromatographic materials, which havebeen subjected to a sanitization process. In an embodiment, theadsorbent materials are selected from the group consisting of syntheticcarbon, anion exchange resins, cation exchange resins, and combinationsthereof.

In an embodiment, the adsorbent material comprises synthetic carbonparticles (SCP) containing micro-, meso- and macropores from porousphenolic resins. As used herein, the term “micropore” refers to a poreswith diameter <2 nm, as measured by nitrogen adsorption and mercuryporosimetry methods and as defined by IUPAC. As used herein, the term“mesopore” refers to pores with diameter from ca. 2 nm to ca. 50 nm, asmeasured by nitrogen adsorption and mercury porosimetry methods and asdefined by IUPAC. As used herein, the term “macropore” refers to poreswith diameters larger than 50 nm, as measured by nitrogen adsorption andmercury porosimetry methods and as defined by IUPAC. In relation to thisdisclosure there are two types of macropores. In macroporous beads theyare located within beads and formed by pore-formers. Their size is50-500 nm, typically 70-200 nm. These macropores are very effective inadsorption of cytokines.

A SCP suitable for use in the present disclosure may have any shapecompatible with the compositions and methodologies disclosed herein. Forexample the shape of the SCP may be that of an irregular granule, a lowangularity shape, spherical (e.g., bead), pellet, minilith, monolith,etc. For simplicity, the present disclosure may refer to the use ofbeads of the SCB however it is to be understood the SCP may be of anysuitable shape. The SCPs may be formed using any suitable methodology toresults in a material having the properties disclosed herein. In anexemplary method for the formation of an SCP, a precursor resinformulation is used which comprises a large proportion of pore former,e.g. 250 parts ethylene glycol or other pore former to 100 parts ofresin-forming components.

Herein a mesoporous resin may be formed by condensing a nucleophiliccomponent which comprises a phenolic compound or a phenol condensationprepolymer with at least one electrophilic cross-linking agent selectedfrom formaldehyde, paraformaldehyde, furfural and hexamethylenetetramine in the presence of a pore-former selected from the groupconsisting of a diol (e.g. ethylene glycol), a diol ether, a cyclicester, a substituted cyclic ester, a substituted linear amide, asubstituted cyclic amide, an amino alcohol and a mixture of any of theabove with water to form a resin. The pore-former is present in anamount effective to impart meso- or macroporosity to the resin (e.g. atleast 120 parts by weight of the pore former being used to dissolve 100parts by weight of the total resin forming components, i.e. nucleophiliccomponent plus electrophilic component), and it is removed from theporous resin after condensation by cascade washing with water or byvacuum drying. The resulting resin may be carbonised by heating in aninert atmosphere to a temperature of at least 600° C. to give a materialhaving a bimodal distribution of pores, the pore structure as estimatedby nitrogen adsorption porosimetry comprising micropores and mesoporesor macropores. The value for the differential of pore volume withrespect to the logarithm of pore radius (dV/dlogR) for the mesopores isgreater than 0.2 for at least some values of pore size in the range20-500 Å. The mesoporous carbon may have a BET surface area of 250-800m²/g without activation. It may be activated by heating it at hightemperature in the presence of carbon dioxide, steam or a mixturethereof, e.g. by heating it in carbon dioxide at above 800° C., or itmay be activated by heating it in air at above 400° C. It may then havesurface areas of up to 2000 m²/g and even higher e.g. 1000-2000 m²/g. Asused herein the term “BET surface area” is determined by the Brunauer,Emmett, and Teller (BET) method according to ASTM D1993-91, see alsoASTM D6556-04.

Resins for making carbonaceous material can be prepared from any of thestarting materials such that the nucleophilic components may comprisephenol, bisphenol A, alkyl phenols e.g. cresol, diphenols e.g.resorcinol and hydroquinione and aminophenols e.g. m-amino-phenol.

It is preferred to use as nucleophilic component a phenolic novolac orother similar oligomeric starting material, which because it is alreadypartly polymerized makes polymerization to the desired resin a lessexothermic and hence more controllable reaction. The preferred novolacshave average molecular weights (AMW) in the range of from 300 to 3000prior to cross-linking (corresponding to a DP with respect to phenol ofabout 3-30). Where novolac resins are used, they may be solids withmelting points in the region of 100° C. Novolac resins of AMW less than2000 and preferably less than 1500 form resins which on carbonisationtend to produce carbons with desired pore size distributions using loweramounts of pore former. Novolacs are thermally stable in that they canbe heated so that they become molten and cooled so that they solidifyrepeatedly without structural change. They are cured on addition ofcross-linking agents and heating. Fully cured resins are infusible andinsoluble. Whilst commercial novolacs are largely produced using phenoland formaldehyde, a variety of modifying reagents can be used at thepre-polymer formation stage to introduce a range of different oxygen andnitrogen functionalities and cross-linking sites. These include but arenot limited to: (a) Dihydric phenols e.g. resorcinol and hydroquinone.Both are more reactive than phenol and can lead to some cross-linking atthe pre-polymer production stage. It is also possible to introduce thesecompounds at the cross-linking stage to provide different cross-linkingpaths. These also increase the oxygen functionality of the resins. (b)Nitrogen containing compounds that are active in polycondensationreactions, such as urea, aromatic (aniline, m-amino phenol) andheteroaromatic (melamine) amines. These allow the introduction ofspecific types of nitrogen functionality into the initial polymer andfinal carbon and influence the development of the mesoporous structureof both the resins and the final carbons. Like hydroquinone andresorcinol, all the nitrogen containing nucleophilic modifying reagentswhich can be used possess two or more active sites and are more reactivein condensation reactions than phenol or novolacs. It means that theyare first to react with primary cross-linking agents forming secondarycross-linking agents in situ.

The nucleophilic component may be provided alone or in association witha polymerization catalyst which may be a weak organic acid miscible withthe novolac and/or soluble in the pore former e.g. salicylic acid,oxalic acid or phthalic acid. The concentration of novolac in the poreformer may be such that when combined with the solution of cross-linkingagent in the same pore former the overall weight ratio of pore former to(novolac+cross-linking agent) is at least 125:100 by weight. The actualratios of novolac:pore former and cross-linking agent:pore former areset according to convenience in operation by the operationalrequirements of a bead production plant and are controlled by theviscosity of the novolac:pore former solution such that it remainspumpable and by the ratio of cross-linking agent:pore former such thatthe cross-linking agent remains in solution throughout the plant.

The cross-linking agent is normally used in an amount of from 5 to 40parts by weight (pbw) per 100 parts by weight of the nucleophiliccomponents e.g. novolac,. It may be, for example, an aldehyde e.g.formaldehyde or furfural, it could be hexamethylenetetramine (hexamine),or hydroxymethylated melamine.

Hexamine is preferably used as cross-linking agent. In embodimentsrequiring a completely cured resin, it is preferably used forcross-linking novolac resin at a proportion of 10 to 25 pbw e.g. about15 to 20 pbw hexamine per 100 pbw of novolac. This ensures formation ofthe solid resin with maximal cross-linking degree and ensures thestability of the mesopore structure during subsequent removal of thepore former.

The pore former also acts as solvent. Thus, the pore former ispreferably used in sufficient quantities to dissolve the components ofthe resin system, the weight ratio of pore former to the totalcomponents of the resin system resin being preferably at least 1.25:1.

The pore former may be, for example, a diol, a diol-ether, a cyclicester, a substituted cyclic or linear amide or an amino alcohol e.g.ethylene glycol, 1,4-butylene glycol, diethylene glycol, triethyleneglycol, .gamma.-butyrolactone, propylene carbonate, dimethylformamide,N-methyl-2-pyrrolidinone and monoethanolamine, ethylene glycol beingpreferred, and where the selection is also limited by the thermalproperties of the solvent as it should not boil or have an excessivevapour pressure at the temperatures used in the curing process.

It is thought that the mechanism of meso- and macropore generation isdue to a phase separation process that occurs during the cross-linkingreaction. In the absence of a pore former, as the linear chains ofpre-polymer undergo cross-linking, their molecular weight initiallyincreases. Residual low molecular weight components become insoluble inthe higher molecular weight regions causing a phase separation intocross-linked high molecular weight domains within the lower molecularweight continuous phase. Further condensation of light components to theoutside of the growing domains occurs until the cross-linked phasebecomes essentially continuous with residual lighter pre-polymer trappedbetween the domains. In the presence of a low level of pore former thepore former is compatible with, and remains within, the cross-linkedresin domains, (e.g., <120 parts/100 parts Novolac for theNovolac-Hexamine-Ethylene Glycol reaction system), whilst the remainderforms a solution with the partially cross-linked polymer between thedomains. In the presence of higher levels of pore former, which exceedthe capacity of the cross-linked resin, the pore former adds to thelight polymer fraction increasing the volume of material in the voidsbetween the domains that gives rise to the mesoporosity and/ormacroporosity. In general, the higher the pore former content, the widerthe mesopores, up to macropores, and the higher the pore volume.

This phase separation mechanism provides a variety of ways ofcontrolling the pore development in the cross-linked resin structures.These include chemical composition and concentration of the pore former;chemical composition and quantity of the cross-linking electrophilicagents, presence, chemical nature and concentration of modifyingnucleophilic agents, chemical composition of phenolic nucleophiliccomponents (phenol, novolac), the presence of water within the solventand concentration of any curing catalyst if present.

Production of the bead form may be by pouring a solution of a partiallycross-linked pre-polymer into a hot liquid such as mineral oilcontaining a dispersing agent and stirring the mixture. The pre-polymersolution forms into beads which are initially liquid and then, as curingproceeds, become solid. The average bead particle size is controlled byseveral process parameters including the stirrer type and speed, the oiltemperature and viscosity, the pre-polymer solution viscosity and volumeratio of the solution to the oil and the mean size can be adjustedbetween 5 and 2000 μm although in practice the larger bead sizes aredifficult to achieve owing to problems with the beads in the stirreddispersion vessel. The beads can then be filtered off from the oil. In apreparative example, industrial novolac resin is mixed with ethyleneglycol at an elevated temperature, mixed with hexamine and heated togive a viscous solution which is poured into mineral oil containing adrying oil, after which the mixture is further heated to effect curing.On completion of curing, the reaction mixture is cooled, after which theresulting porous resin is filtered off, and washed with hot water toremove pore former and a small amount of low molecular weight polymer.The cured beads are carbonized to porous carbon beads which have a porestructure as indicated above, and may be activated as indicated above.It is stated that the beads can be produced with a narrow particle sizedistribution e.g. with a D90.D10 of better than 10 and preferably betterthan 5. However, the bead size distribution that can be achieved inpractice in stirred tank reactors is relatively wide, and the more theprocess is scaled up the worse the homogeneity of the mixing regime andhence the particle size distribution becomes wider.

Discrete solid beads of polymeric material e.g. phenolic resin having aporous structure may be formed, which process may produce resin beads onan industrial scale without aggregates of resin building up speedily andinterrupting production. The process comprises the steps of: (a)combining a stream of a polymerizable liquid precursor e.g. a novolacand hexamine as cross-linking agent dissolved in a first polar organicliquid e.g. ethylene glycol with a stream of a liquid suspension mediumwhich is a second non-polar organic liquid with which the liquidprecursor is substantially or completely immiscible e.g. transformer oilcontaining a drying oil; (b) mixing the combined stream to disperse thepolymerizable liquid precursor as droplets in the suspension medium e.g.using an in-line static mixer; (c) allowing the droplets to polymerisein a laminar flow of the suspension medium so as to form discrete solidbeads that cannot agglomerate; and (d) recovering the beads from thesuspension medium.

For bead production, the pore former comprises a polar organic liquide.g. ethylene glycol chosen in combination with dispersion medium whichis a non-polar organic liquid so as to form a mainly or whollyimmiscible combination, the greater the incompatibility between the poreformer which forms the dispersed phase and the dispersion medium, theless pore former becomes extracted into the dispersion medium. The poreformer desirably has a greater density than the dispersion medium withwhich it is intended to be used so that droplets of the pore formercontaining dissolved resin-forming components will pass down a columnmore rapidly than a descending flow of dispersion medium therein. Bothprotic and aprotic solvents of different classes of organic compoundsmatch these requirements and can be used as pore formers, bothindividually and in mixtures. In addition to dissolving the reactivecomponents and any catalyst, the pore former should also, in the case ofphenolic resins, be compatible with water and/or other minorcondensation products (e.g. ammonia) which are formed by elimination aspolymerization proceeds, and the pore former is preferably highlymiscible with water so that it can be readily removed from thepolymerized resin beads by washing.

The dispersion medium is a liquid which can be heated to the temperatureat which curing is carried out e.g. to 160° C. without boiling atambient pressure and without decomposition and which is immiscible withethylene glycol and with the dissolved components therein. It may behydrocarbon-based transformer oil which is a refined mineral oil and isa by-product of the distillation of petroleum. It may be composedprincipally of C.15-C.40 alkanes and cycloalkanes, have a density of0.8-0.9 depending upon grade and have a boiling point at ambientpressure of 260-330° C., also depending upon grade. Transformer oil hasa viscosity of about 0.5 poise at 150° C. which is a typical curetemperature. Transformer oil or other dispersion medium may be used involumes 3-10 times the volume of the combined streams of nucleophilicprecursor and crosslinking agent e.g. about 5 times.

Preferred dispersing agents which are dissolved in the dispersion mediumbefore that medium is contacted with the reaction mixture to bedispersed therein to retard droplet coalescence are either sold asdrying oils e.g. Danish oil or are produced by partially oxidizingnaturally occurring precursors such as tung oil, linseed oil etc. Thedispersing agents are consumed as the process proceeds, so that if thedispersion medium is recycled, dispersing agent in the recycled oilstream should be replenished. The dispersing agent is convenientlysupplied as a stream in solution in the dispersion medium e.g.transformer oil and e.g. in an amount of 5-10% v/v where Danish oil isused which contains a low concentration of the active component to givefinal concentration of the dispersant in the dispersion medium 0.2-1%v/v. Higher dispersant concentrations would be used in the case ofoxidised vegetable oils.

The resin beads formed as described above may be carbonised andoptionally activated. For example, carbonization and activation maycomprise supplying the material to an externally fired rotary kilnmaintained at carbonizing and activating temperatures, the kiln having adownward slope to progress the material as it rotates, the kiln havingan atmosphere substantially free of oxygen provided by a counter-currentof steam or carbon dioxide, and annular weirs being provided atintervals along the kiln to control progress of the material. In anembodiment, a SCP suitable for use in the present disclosure ischaracterized by a microporous/macroporous structure.

In an embodiment, the SCP has a macroporous pore size of from about 75μm to about 1000 μm, alternatively the SCP has a macroporous size offrom about 100 μm to about 750 μm, or alternatively from about 100 μm toabout 500 μm. Herein an SCP suitable for use in the present disclosuremay comprise an SCP having at least two pore size distribution such thatthe SCP is a mixture of carbon beads having at least two distributionsof macroporous pore sizes. In an embodiment, the SCP may comprise afirst population having a macroporous pore size denoted x and a secondpopulation having a macroporous pore size y where the SCP provides amixture having a ratio of x/y of about 1; alternatively about 5,alternatively about 10, alternatively about 20; alternatively about 50,or alternatively about 100. In some embodiments, the SCP comprises amixture of two populations wherein the pore size of the first populationis approximately twice the pore size of the second population. In someembodiments, the SCP comprises a mixture of three populations where thepore size of a first population is approximately twice the pore size ofthe second population and the pore size of the third population isapproximately two and a half times the pore size of the secondpopulation.

In an embodiment, the adsorbent material comprises an ion exchange resin(IER). Herein an IER refers to an insoluble matrix fabricated from asubstrate and functionalized with a fixed ion and a mobile counterion.The IER retards ions on the surface of the material with the concomitantrelease of the mobile counterion. IERs can also be described asinsoluble polymers that contain ionizable groups distributed regularlyalong the polymer backbone. As a consequence, any counter ion associatedwith the ion exchange resin is ionically bound to the ion exchange resinand physically separated from the surrounding fluid.

In an embodiment, an IER suitable for use in the present disclosure hasa bead size ranging from about 40 μm to about 1000 μm, alternativelyfrom about 40 μm to about 750 μm, or alternatively from about 100 μm toabout 500 μm.

In an embodiment, the IER is an anion exchange resin. Herein “anionexchange resin” refers to an ion exchange resin with covalently boundpositively charged groups, such as quaternary amino groups and mobilenegatively charged groups. The term “anion exchange resin” is intendedto encompass strong base anion exchange resins (SBA), weak base anionexchange resins (WBA) and related anionic functional resins, of eitherthe gellular or macroporous type containing quaternary ammoniumfunctionality (chloride, hydroxide or carbonate forms), dialkylamino orsubstituted dialkylamino functionality (free base or acid salt form),and aminoalkylphosphonate or iminodiacetate functionality, respectively.Examples of commercially available anion exchange resins suitable foruse in the present disclosure include without limitation those soldunder the tradename of DEAE, QAE, and UNOSphere. In an embodiment, theanion exchange resin comprises UNOSphere Q Media.

In an embodiment, the IER is a cation exchange resin. The cationexchange resin of the present disclosure may be strongly or weeklyacidic and have a variety of functional groups, e.g., weakly acidic typeof resin containing carboxylic acid group, or strongly acidic type ofresin containing sulfonic functional groups. Generally, the carboxylicfunctional groups may be derived from polymers or copolymers ofmethacrylic acid or polymethacrylic acid and the sulfonic functionalgroups may generally be derived from polymers or copolymers of styreneand divinylbenzene. Other polymeric matrices, organic ion exchangematrices or inorganic ion exchange matrices may be used as suitable ionexchange resins, e.g., methacrylic, acrylic and phenol formaldehyde. Forexample, cation exchange resins suitable for use in the presentdisclosure include without limitation AMBERLITE and UNOSphere S Media.AMBERLITE is described by the manufacturer as gel-type divinylbenzenesulfonic acid cation exchange resin that swells in water.

In an embodiment, an adsorbent material suitable for use in the presentdisclosure has been subjected to a sanitization process. Herein thesanitization process refers to a method of treating the adsorbentmaterials in order to (i) remove pathogens; (ii) reduce the amount offine particulates and leachables; (iii) reduce the amount of trapped airand (iv) sterilize the materials. Adsorbent materials that have beensubjected to the sanitization process disclosed herein are considered tohave been converted from an industrial grade material to apharmaceutical grade material with a concomitant increase inhemocompatability.

In an embodiment, a method for sanitization of a SCP of the typedisclosed herein comprises a dry heat treatment to produce aheat-treated SCP. Dry heat treatment of the SCP may be carried out at atemperature at equal to or greater than about 180° C. for a time periodequal to or greater than about 4 hours, alternatively at a temperatureof equal to or greater than about 200° C. for a time period of equal toor greater than about 1 hour, or alternatively at a temperature of 250°C. for a time period of equal to or greater than about 30 min. Dry heattreatment of the SCP may function to reduce the bioburden of thematerial and particularly the amount of pathogenic (e.g., bacteria,viruses, fungi, etc . . . ) and pyrogenic (e.g., endotoxin) substancesassociated with the SCP. For example, the total amount of pathogenicsubstances associated with the heat-treated SCP may be reduced bygreater than about 50%, alternatively greater than about 90%,alternatively greater than about 91%, alternatively greater than about92%, alternatively greater than about 93%, alternatively greater thanabout 94%, alternatively greater than about 95%, alternatively greaterthan about 96%, alternatively greater than about 97%, alternativelygreater than about 98%, alternatively greater than about 99%, oralternatively about 100% when compared to the SCP.

In an embodiment, the bioburden of the SCP is reduced by about 100%through the use of a dry heat treatment. Alternatively, the bioburden ofthe SCP is reduced through the use of any suitable methodologycompatible with the SCP and the other components of the presentdisclosure. In some embodiments, the bioburden of the SCP is reduced by100% utilizing methodologies consistent with jurisdictional guidelinesfor the sanitization of materials that will contact mammalian blood andproduce a product that will be subsequently utilized in mammals.

In an embodiment, a method for sanitization further comprises theremoval of fine particulates and leachables from the heat-treated SCP.Herein particulates smaller than about 30 microns are referred to as“fines” while “leachables” describe the organic compounds that can beeluted from the adsorbent material (e.g., heat-treated SCP) in thepresence/absence of an applied sample. In an embodiment, removal of thefine particulates and leachables from the heat-treated SCP comprisescontacting the heat-treated SCP with water, removing water from theheat-treated SCP to produce a washed SCP, contacting the washed SCP witha salt solution to produce a modified SCP and removing the salt solutionfrom the modified SCP to produce a processed SCP. The heat-treated SCPmay be contacted with from about 4 volumes to about 10 volumes of water,alternatively from about 5 volumes to about 10 volumes of water oralternatively from about 6 volumes to about 8 volumes of water.Contacting of the adsorbent material with a substance may be carried outin any suitable vessel. For example, the adsorbent material (e.g.,heat-treated SCP) may be positioned within a cartridge or column tofacilitate contacting of the adsorbent material with one or moresubstances of the type disclosed herein. For example, the washed SCP maybe contacted with a salt solution comprising a sodium chloride salt at aconcentration of 3 g/dL. The washed SCP may be contacted with from about4 volumes to about 10 volumes of salt solution based on the total volumeof the SCP, alternatively from about 6 volumes to about 10 volumes ofsalt solution or alternatively from about 6 volumes to about 8 volumesof salt solution. It is contemplated that other salt solutions providingsimilar pH and osmolarity, such as known to the ordinarily skilledartisan and compatible with the other methods and compositions of thepresent disclosure, may be employed to facilitate the removal of fineparticulates and leachables from the SCP.

For either the removal of water to produce a washed SCP or the removalof salt to produce a processed SCP, the removal may be effected usingany suitable methodology. For example, the removal of fine particulatesand leachables may be carried out by placing the adsorbent material in acolumn which may be allowed to drain under gravity until no furtherfiltrate is detected in order to remove the water and/or salt solution.In some embodiments, the adsorbent material may be subjected to aplurality of processes for the removal of fine particulates andleachables. Further, in some embodiments, the solution produced bycontacting the adsorbent material with water and/or a salt solution maybe analyzed to determine the amount of fine particulates and/orleachables removed following contact. Such determinations may be madeand the process for removal of fine particulates and/or leachablesrepeated until some user and/or process desired level of fineparticulates and/or leachables is achieved.

In an embodiment, a method for sanitization further comprises dewateringthe processed SCP. Water present with the adsorbent material has thetendency to separate from the material resulting in compaction and areduction in flow properties. De-watering is the process of removingextraneous fluid (typically water or aqueous solutions) from wet orslurried particles without removing fluid in the particles (i.e.,prevent evaporative drying of the particles). Herein “extraneous” meansany fluid outside the particles. Therefore any fluid absorbed into thepolymer matrix or present in the pores is not considered extraneous.

Any suitable methodology may be employed for the dewatering of theprocessed SCP. Examples of methodologies suitable for use in dewateringthe processed SCP include without limitation the passage of air throughthe particles. The resultant material is referred to as the dewateredSCP. In an embodiment, dewatering of the processed SCP is carried outusing a dewatering apparatus.

In an embodiment, a method for sanitization further comprises asepticprocessing of the dewatered SCP, also referred to as sterile fill andsterilization to produce a sanitized SCP. Sterility may be achievedusing any suitable methodology. For example sterile processing mayinclude the use of clean rooms, bacteria retaining filters, and dry orsteam heat. In an embodiment, aseptic processing of the dewatered SCPcomprises terminal sterilization by autoclaving (e.g., at 121° C., 15psi for 30 min), gas sterilization, e-beam sterilization, gammaradiation, or combinations thereof

In an embodiment, the adsorbent material is an IER (e.g., anion exchangeresin) and a method for sanitization of an IER comprises the removal offine particulates. Removal of the fine particulates from the IER maycomprise contacting the IER with water, removing water from the IER toproduce a washed IER, contacting the washed IER with a salt solution toproduce a modified IER and removing the salt solution from the modifiedIER to produce a processed IER. The IER may be contacted with from about4 volumes to about 10 volumes of water based on the total volume of theIER, alternatively from about 6 volumes to about 10 volumes of water oralternatively from about 6 volumes to about 8 volumes of water.Contacting of the adsorbent material with a substance may be carried outin any suitable vessel. For example, the adsorbent material (e.g., IER)may be positioned within a cartridge or column to facilitate contactingof the adsorbent material with one or more substances of the typedisclosed herein. In an embodiment, the washed IER is contacted with asalt solution comprising for example 0.9% NaCl in water. It iscontemplated that other salt solutions, such as known to the ordinarilyskilled artisan providing similar pH and osmolarity, and compatible withthe other methods and compositions of the present disclosure, may beemployed to facilitate the removal of fine particulates and leachablesfrom the IER. The washed IER may be contacted with from about 2 volumesto about 8 volumes of salt solution based on the total volume of IER,alternatively from about 4 volumes to about 8 volumes of salt solutionor alternatively from about 6 volumes to about 8 volumes of saltsolution. For either the removal of water to produce a washed IER or theremoval of salt to produce a processed IER, the removal may be effectedusing any suitable methodology. For example, the removal of fineparticulates may be carried out by placing the adsorbent material in acolumn which may be allowed to drain under gravity until no furtherfiltrate is detected in order to remove the water and/or salt solution.In some embodiments, the adsorbent material may be subjected to aplurality of processes for the removal of fine particulates. Further, insome embodiments, the solution produced by contacting the adsorbentmaterial with water and/or a salt solution may be analyzed to determinethe amount of fine particulates. Such determinations may be made and theprocess for removal of fine particulates repeated until some user and/orprocess desired level of fine particulates is achieved.

In an embodiment, a method for sanitization further comprisesautoclaving the processed IER. Autoclaving of the processed IER may becarried out at a temperature of equal to or greater than about 121° C.for a period of time equal to or greater than about 30 min,alternatively equal to or greater than about 60 min, or alternativelyfor a period of time from about 30 min to about 60 min. The resultantmaterial is termed an autoclaved IER.

The autoclaved IER may be further processed by undergoing a high pHtreatment. For example, the autoclaved IER may be contacted with anabout 0.5 M to about 2M NaOH solution for a period of time equal to orless than about 24 hours It is contemplated that other basic solutionsproviding the pH characteristics of a 0.5 M-2M NaOH solution andcompatible with the other aspects of this disclosure may be employed forhigh pH treatment of the IER. The resultant material is termed apH-treated IER. Autoclaving of the processed IER may function to reducethe bioburden of the material and particularly the amount of pathogenic(e.g., bacteria, viruses, fungi, etc . . . ) and pyrogenic (e.g.,endotoxin) substances associated with the processed IER. For example,the total amount of pathogenic substances associated with the autoclavedIER may be reduced by greater than about 50%, alternatively greater thanabout 90%, alternatively greater than about 91%, alternatively greaterthan about 92%, alternatively greater than about 93%, alternativelygreater than about 94%, alternatively greater than about 95%,alternatively greater than about 96%, alternatively greater than about97%, alternatively greater than about 98%, alternatively greater thanabout 99%, or alternatively about 100% when compared to the IER. In anembodiment, the bioburden of the IER is reduced by about 100% throughthe use of methodologies disclosed herein. Alternatively, the bioburdenof the IER is reduced by about 100% through the use of any suitablemethodology compatible with the IER and the other components of thepresent disclosure. In some embodiments, the bioburden of the IER isreduced by 100% utilizing methodologies consistent with jurisdictionalguidelines for the sanitization of materials that will contact mammalianblood and produce a product that will be subsequently utilized inmammals.

In an embodiment, a method for sanitization further comprises thechromatographic removal of base and leachables from the pH-treated IER.For example, the IER may be disposed within a column and contacted withsufficient volumes of a low concentration salt solution to provide anfiltrate having a neutral pH. In an embodiment the IER may be washedwith a 3% NaCl solution until the filtrate has a pH ranging from about7.4 to about 7.6. It is contemplated that other salt solutions, such asknown to the ordinarily skilled artisan and compatible with the othermethods and compositions of the present disclosure, may be employed tofacilitate the removal of bases and leachables from the IER. Theresultant material is termed a modified IER.

In an embodiment, a method for sanitization further comprises dewateringthe modified IER to produce a dewatered IER. Herein dewatering refers tothe removal of water from the adsorbent materials. Water present withthe adsorbent material has the tendency to separate from the materialresulting in compaction and a reduction in flow properties. Any suitablemethodology may be employed for the dewatering of the IER. Examples ofmethodologies suitable for use in dewatering the IER are describedherein with regards to dewatering of the SCP.

In an embodiment, a method for sanitization further comprises asepticprocessing of the dewatered IER, also referred to as sterile fill andsterilization to produce a sanitized IER. Sterility may be achievedusing any suitable methodology. For example sterile processing mayinclude the use of clean rooms, bacteria retaining filters, dry or steamheat, terminal sterilization by autoclaving at 121° C., 15 psi for 30min, gas sterilization, e-beam sterilization, gamma radiation, orcombinations thereof. In some embodiments, methods for sanitization ofthe SCP, IER, or both do not comprise or alternatively exclude asepticprocessing.

The sanitization process disclosed herein may be performed using anysuitable equipment and/or having the adsorbent material disposed withinany suitable vessel for performing one or more steps of the sanitizationprocess. In an embodiment, the adsorbent material is disposed within acolumn and the sanitization process is carried out without transfer ofthe adsorbent material to another container or vessel. In suchembodiments, the adsorbent material is subjected tosanitization-in-place (SIP).

In an embodiment, adsorbent materials subjected to a sanitizationprocess, both of the type disclosed herein, are characterized by abioburden maximum of 20 endotoxin units (EU)/blood/plasma contactingdevice and 2.15 endotoxin units (EU)/CSF contacting device as determinedusing any suitable methodology such as the Limulus amebocyte lysatetest. In an embodiment, adsorbent materials subjected to a sanitizationprocess, both of the type disclosed herein, are characterized as fineparticulates free which are defined herein as having less than about 1%fine particulates as determined by laser diffraction. Methodologies ofthe type disclosed herein may result in in adsorbent materials havingless than about 0.5%, 0.1% or undetectable amounts of fine particulates.In an embodiment, adsorbent materials subjected to a sanitizationprocess, both of the type disclosed herein, are characterized asleachables free which are defined herein as having less than about 1%leachables as determined spectrophotometrically in the wavelength rangeof 205 nm to 340 nm. Methodologies of the type disclosed herein mayresult in in adsorbent materials having less than about 0.5%, 0.1% orundetectable amounts of leachables. Such materials are collectivelyreferred to herein as sanitized adsorbent materials (SAM).

In an embodiment, the SAM is sanitized in accordance with the UnitedStates Food and Drug Administration Code of Federal Regulations Title 21section 876.5870 for the regulation of Sorbent and Hemoperfusionsystems.

In an embodiment, SAMs suitable for use in the present disclosure (e.g.,in the columns of apparatus 300) are further subjected to contact with acompatibilizer which functions to coat at least a portion of the surfacearea of the SAM. Herein a compatibilizer refers to a substance thatfunctions to increase the biocompatibility of the SAM with biologicalfluids and may aid in decreasing the binding of non-target molecules tothe SAM. In an embodiment, the compatibilizer comprises apolysaccharide, a glucan, albumin, mannitol, a starch, or combinationsthereof.

In an embodiment, the compatibilizer comprises dextran. Dextrans,representations are depicted in Formula 1, are polysaccharides having alinear backbone of a-linked D-glucopyranosyl repeating units. In anembodiment, a dextran suitable for use in the present disclosure has anaverage molecular weight ranging from about 1 kDa to about 500 kDa,alternatively from about 1 kDa to about 70 kDa, alternatively from about1 kDa to about 40 kDa, or alternatively from about 40 kDa to about 70kDa. Nonlimiting examples of compatibilizers suitable for use in thepresent disclosure include DEXTRAN-1, DEXTRAN-40 and DEXTRAN-70commercially available from Hospira Inc.

In an embodiment, the compatibilizer comprises hydroxyethyl starch.Hydroxyethyl starch, depicted in Formula II, is a nonionic starchderivative that is commonly used as a volume expander in a type ofintravenous therapy that has the function of providing volume for thecirculatory system.

In an embodiment the compatiblizer comprises a mixture of albumin andmannitol. Serum albumin is the main protein of human blood plasma whoseprimary function is to regulate the colloidal osmotic pressure of blood.Mannitol, (2R,3R,4R,5R)-Hexan-1,2,3,4,5,6-hexol, is a sugar alcohol,which can function an Osmotic Diuretic. The weight ratio of albumin tomannitol in the compatibilizer may range from 20:1 to 1:1, alternativelyfrom 18:1 to 1:1, or alternatively from 15:1 to 10:1.

Without wishing to be limited by theory, the compatibilizer (e.g.,dextran) may function to prime the extracorporeal circuit (i.e.,apparatus having columns containing the adsorbent materials) and maylessen complications by blocking the initial exposure of bloodcomponents and plasma to foreign surfaces while maintaining a higherlevel of colloid oncotic pressure. In an embodiment, the compatibilizeris dextran 40 which may function in (i) preventing shear-induced finesformation via a lubrication effect; (ii) serving as a priming agent forthe extracorporeal circuit assembled with the blood separator and theadsorbing device to prevent activation of plasma and other bloodcomponents following early primary exposure; and (iii) modulatingsorbing capacity of porous sorbents such as syntheticmesoporous/microporous carbon. For example, the adsorbents packed intocolumns as components of an apparatus of the type disclosed herein,during storage/distribution can be exposed to relatively high shearstresses which can be a continuous source of particulates while dextranmay prevent fines formation by lubrication at any shear condition.

SAMs suitable for use in the present disclosure may be contacted withthe compatibilizer using any suitable methodology. In an embodiment, thecompatibilizer is dextran which may be formulated as a solution suitablefor use in the present disclosure having from about 1 weight percent(wt. %) dextran about 10 wt. % dextran, alternatively from about 2 wt. %to about 9 wt. % or alternatively from about 3 wt. % to about 7 wt. %.In an embodiment, the compatibilizer is hydroxyethyl starch which may beformulated as a solution suitable for use in the present disclosurehaving from about 1 wt. % to about 6 wt. % hydroxyethyl starch,alternatively from about 1.5 wt. % to about 6 wt. % hydroxyethyl starchor alternatively from about 2 wt. % to about 6 wt. % hydroxyethylstarch. The resultant compatibilized SAM (C-SAM) may be characterized bythe formation of a coating of the compatibilizer on the particles of theSAM such that the coating covers greater than about 50% of theparticle's surface; alternatively, greater than about 60%, 70%, 80% or90% of the particle's surface.

In an embodiment, C-SAMs are introduced to the columns of apparatus 300.For example, the apparatus may be operated having sanitized SCP incolumn A (referring to FIG. 1A, column 310), a mixture of a sanitizedSCP and a sanitized anionic exchange resin in column B (referring toFIG. 1, column 350) and a mixture of a SCP and a cationic exchange resinin column C (referring to FIG. 1, column 370). In an embodiment, thedisclosed methodology comprises an extracorporeal device of the typedepicted as apparatus 300 wherein bodily fluids (e.g., blood) obtainedfrom a subject infected with a VISE (e.g., Ebola virus) or suspected ofbeing infected with a VISE are contacted with the materials housed inthe depicted columns in a sequence consisting essentially of contactingwith a sanitized compatibilized SCP that is disposed within a firstcolumn (e.g., column A 310) to form a first filtrate that is introducedto a second column (e.g., column B 350) and contacted with a mixture ofa sanitized compatibilized SCP and a sanitized compatibilized anionexchange resin to form a second filtrate. The second filtrate maysubsequently introduced to a third column (e.g., column C 370) andcontacted with a mixture of a sanitized compatibilized SCP and asanitized compatibilized cation exchange resin to form a third filtrate.In an embodiment, a method of treating a subject infected with the VISE(e.g., Ebola virus) or suspected of being infected with the VISE (e.g.,Ebola virus) comprises administering to the subject at least a portionof the third filtrate. In some embodiments, the third filtrate may befurther processed by the addition of one or more agents that function toameliorate the symptoms of the VISE.

In an embodiment, columns having both a sanitized, compatibilized SCPand a sanitized, compatibilized anion exchange resin disposed thereinmay have the ratio of sanitized compatibilized SCP to sanitizedcompatibilized anion exchange of from about 0.1:100 to about 100:0.1,alternatively from about 1:100 to about 100:1, or alternatively fromabout 10:100 to about 100:10, For example, columns having both asanitized, compatibilized SCP and a sanitized, compatibilized anionexchange resin disposed therein may have the ratio of sanitizedcompatibilized SCP to sanitized compatibilized anion exchange of about1:1, alternatively about 100:1, alternatively about 1:50, alternativelyabout 50:1, alternatively about 1:25, or alternatively about 25:1. Inanother embodiment, columns having both a sanitized, compatibilized SCPand a sanitized, compatibilized cation exchange resin disposed thereinmay have the ratio of sanitized compatibilized SCP to sanitizedcompatibilized cation exchange of from about 0.1:100 to about 100:0.1,alternatively from about 1:100 to about 100:1, or alternatively fromabout 10:100 to about 100:10, For example, columns having both asanitized compatibilized SCP and a sanitized compatibilized cationexchange resin disposed therein may have the ratio of sanitizedcompatibilized SCP to sanitized compatibilized cation exchange resinrange from of about 1:1, alternatively about 100:1, alternatively about1:50, alternatively about 50:1, alternatively about 1:25, oralternatively about 25:1.

In an embodiment, columns having both a sanitized, compatibilized SCPand a sanitized, compatibilized anion exchange resin disposed thereinmay have the amount of sanitized compatibilized SCP to sanitizedcompatibilized anion exchange be from about 1 wt. % SCP to about 99 wt.% anion exchange resin based on the total dry weight of adsorbentmaterials, alternatively form about 10 wt. % SCP to about 90 wt. % anionexchange resin, alternatively from about 20 wt. % SCP to about 80 wt. %anion exchange resin, alternatively from about 30 wt. % SCP to about 70wt. % anion exchange resin, alternatively from about 50 wt. % SCP toabout 50 wt. % anion exchange resin, alternatively from about 75 wt. %SCP to about 25 wt. % anion exchange resin, alternatively from about 80wt. % SCP to about 20 wt. % anion exchange resin, alternatively fromabout 90 wt. % SCP to about 10 wt. % anion exchange resin, oralternatively from about 99 wt. % SCP to about 1 wt. % anion exchangeresin. In another embodiment, columns having both a sanitizedcompatibilized SCP and a sanitized compatibilized cation exchange resindisposed therein may have the amount of sanitized compatibilized SCP tosanitized compatibilized cation exchange resin be from about 1 wt. % SCPto 99 wt. % cation exchange resin based on the total weight of adsorbentmaterials, alternatively from about 10 wt. % SCP to about 90 wt. %cation exchange resin, alternatively from about 20 wt. % SCP to about 80wt. % cation exchange resin, alternatively from about 30 wt. % SCP toabout 70 wt. % cation exchange resin, alternatively from about 50 wt. %SCP to about 50 wt. % cation exchange resin, alternatively from about 75wt. % SCP to about 25 wt. % cation exchange resin, alternatively fromabout 80 wt. % SCP to about 20 wt. % cation exchange resin,alternatively from about 90 wt. % SCP to about about 10 wt. % cationexchange resin, or alternatively from about 99 wt. % SCP to about 1 wt.% cation exchange resin.

In an embodiment, a subject suffering from a VISE or suspected ofsuffering from a VISE may be treated using the methodologies disclosedherein. For example, the subject may be placed in fluid communicationwith an extracorporeal apparatus of the type disclosed herein so as toallow bodily fluid (e.g., blood) of the subject to flow into an inletport of the device. The extracorporeal apparatus may have columnssituated in the apparatus to afford contact of the incoming bodily fluidwith at least a first column having a sanitized SCP disposed therein toproduce a first filtrate. The first filtrate may then be introduced to asecond column having a mixture of a sanitized compatibilized SCP and asanitized compatibilized anionic resin to produce a second filtrate. Thesecond filtrate may be subsequently introduced to a third columncomprising a sanitized compatibilized SCP and a sanitized compatibilizedcationic resin to produce a third filtrate which may be returned to thesubject.

In an embodiment, treatment of a subject suffering from a VISE orsuspected of suffering from a VISE may result in a reduction in thelevel of disease mediators present in the bodily fluid (e.g., blood) ofthe subject. The presence of an elevated level of these diseasemediators may result in hypercytokinemia where a number ofpro-inflammatory cytokines and other pro-inflammatory substances arereleased by the immune cells within the body. A pro-inflammatorycytokine or a pro-inflammatory mediator is an immuno-regulatory cytokinethat favors inflammation. Pro-inflammatory cytokines that are generallyresponsible for early immune responses include IL-1, IL-6, and TNFα.IL-1, IL-6, and TNF-α are also considered endogenous pyrogens as theycontribute to increasing body temperature. Other examples ofpro-inflammatory cytokines or pro-inflammatory mediators include IL-8,IL-4, L-11, IL-12, IL-18, GM-CSF, IFN-γ, TGF-β, leukemia, inhibitoryfactors (LIF), oncostatin M (OSM), and a variety of chemokines thatattract inflammatory cells.

A pro-inflammatory cytokine generally up-regulates or increases thesynthesis of secondary pro-inflammatory mediators and otherpro-inflammatory cytokines by immune cells. In addition,pro-inflammatory cytokines can stimulate production of acute phaseproteins that mediate inflammation and attract inflammatory cells. IL-1is an example of a pro-inflammatory cytokine. IL-1 is a soluble proteinhaving a mass of approximately 17 kilo-Daltons (kD). IL-1 is produced bya variety of cells, for example macrophages, white blood cells,lymphocytes, monocytes, dendritic cells, and accessory cells that areinvolved in activation of T-lymphocytes and B-lymphocytes. IL-1 istypically released by such cells during an immune response. IL-1 isgenerally considered to be a pro-inflammatory cytokine. Pro-inflammatorycytokines generally refer to immunoregulatory cytokines that favorinflammation.

The original members of the IL-1 superfamily are interleukin-1 alpha(IL-1α), interleukin-1 beta (IL-β), and interleukin-1 receptorantagonist (IL-1RA) Both IL-1α and IL-1β play important roles in theinflammatory response of the body against pathogens or infection. BothIL-1α and IL-1β recognize a same IL-1 receptor and perform similarbiological functions. IL-1α is predominantly a cell-associated moleculewhereas IL-1β is generally a secreted molecule.

IL-1 is produced during immune responses. A common function of IL-1(e.g. IL-1α and IL-1β) is an increasing of expression of adhesionfactors on endothelial cells to enable transmigration of leukocytes(which are immune cells that fight pathogens) to sites of infection. Inaddition, IL-1 stimulates the hypothalamus thermoregulatory center tocause an increase in body temperature (i.e. a fever). The increased bodytemperature helps the body's immune system to fight pathogens orinfection within the body.

TNF-α is also an important pro-inflammatory cytokine. TNF-α is involvedin systemic inflammation and works in tandem with a variety of othercytokines to stimulate the acute phase immune reaction. TNF-α is capableof inducing apoptotic cell death, induce inflammation, as well asinhibit tumorigenesis and viral replication. TNF-α and IL-1 commonlyworks simultaneously and synergistically in stimulating and sustaininginflammation within the body.

In an embodiment, the methods disclosed herein comprise extracorporealexposure of the blood of the subjects in need thereof, to sanitizedcompatibilized adsorbent materials for a time sufficient to reduce thelevel of VISE-related disease mediators to less than about 50% of theamount present in the untreated bodily fluid, alternatively less thanabout 75%, or alternatively less than about 95%. For example the presentmethodologies may be effective in the removal from a bodily fluid (e.g.,blood) of one or more substances associated with suppression of thesubject's immune system (e.g., IL-18, IFN-γ, TNF-α, IL-1β), systemicinflammatory response syndrome (SIRS), (e.g., TNF-α, IL-1β, IL-6, IL-10,MCP-1, MCSF, MIP-1α), hypotension (e.g., NO) that leads to MOF, andcompromise of vascular integrity (e.g., C3a, C5a, histamine) causinginternal and external bleeding. Additional disease mediators arepresented throughout the examples of the present disclosure.

TABLE 1 Desig- Name Also Known As nated Interleukin 18 Interferon-gammainducing factor IL-18 Interferon gamma IFN-γ Tumor necrosis Cachexin,cachectin TNF-α factor alpha Interleukin 6 placental protein 12 (PP12)IL-6 Interleukin-1 beta IL1-β Interleukin 10 Human cytokine synthesisinhibitory IL-10 factor (CSIF) Monocyte chemotactic Chemokine (C-Cmotif) ligand 2 MCP-1 protein 1 (CCL2) Small inducible cytokine A2macrophage colony colony stimulating factor 1 (CSF1) M-CSF stimulatingfactor Macrophage Chemokine (C-C) motif ligand 3 MIP1α inflammatory(CCL3) protein Nitric oxide NO C3a Formed by cleavage of complementcomponent 3 C5a Formed by cleavage of complement component C5 histamine2-(1H-imidazol-4-yl)ethanamine

The effect of treatment of a VISE in terms of progression of the diseasestate may be monitored by any suitable methodology. For example, thelevel and amounts of one or more of the disclosed disease mediators(e.g., Table 1) in the blood of infected individuals may be determinedand monitored during the course of treatment with the methodologiesdisclosed herein. In some embodiments, removal of VISE disease mediatorsmay be determined by immunological methods such as enzyme-linkedimmunoassay (ELISA) and spectrophotometry, or combination thereof. Thesubject's improvement may also be evaluated clinically utilizing metricssuch as but not limited to body temperature, hemodynamic parameters(blood pressure, pulse pressure, EKG) and general signs of improvement.For example, the compositions and methodologies disclosed herein mayresult in the reduction or inhibition the SIRS that develops due to theproduction of a “cytokine storm.” Reduction or inhibition of the SIRSmay be assessed by determination of the level of C-Reactive Protein(CRP) in the subject. In another embodiment, the compositions andmethodologies disclosed herein may result in the improvement inVISE-associated hypotension. In another embodiment the compositions andmethodologies disclosed herein may result in the improvement of thegeneral health status of the VISE-infected subject as assessed bymeasurements of organ function such as but not limited to respiratoryfunction, kidney function, and liver function. In another embodiment,the compositions and methodologies disclosed herein may result in theimprovement of the immunological functioning of the VISE-infectedsubject as assessed by standard immunofunction assays such as a completeblood cell count with differential. In another embodiment, thecompositions and methodologies disclosed herein may result in theimprovement of the general health of the VISE-infected subject asassessed by decreases in the morbidity of a subject population andincreased incidence of subject survival.

The present disclosure contemplates focusing on the pathophysiology ofVISE infection allows deploying effective methods of treatment. Thisdisclosure relates generally to extracorporeal cleansing of blood of asubject with a VISE. More specifically, it relates to the removal ofVISE-associated disease mediators, permitting an infected subject torecover by regaining immune system capacity against the virus.

EXAMPLES

The subject matter of the present disclosure having been generallydescribed, the following examples are given as particular embodiments ofthe disclosure and to demonstrate the practice and advantages thereof.It is understood that the examples are given by way of illustration andare not intended to limit the specification or the claims to follow inany manner.

Example One

Removal of EVD-related Mediators by Mesorporous/Microporous SyntheticCarbon Beads From Human Fresh Frozen Plasma.

Two different synthetic carbon bead materials:—CarbonS-250/500TE7/20-4W-40C and —Carbon S-125/250-TE7/20-4W-50C wereequilibrated prior to testing, and evaluated for the removal of a rangeof target molecules from certified human fresh frozen plasma (FFP).Prior to treatment, FFP (Lot No. 9247350, Sera Care Life Sciences,Milford, Mass.) was spiked with human cytokines (QIAGEN, Inc., Valencia,Calif.) at the following concentrations (pg/mL): TNF-α (100-225); IL-1beta (70-110); IL-6 (80-115); IL-8 (225-475); IL-10 (85-125); TGF-beta 1(450-1,450); INF-gamma (130-520); MCP-1 (120-160); and CRP (10-15 mg/L).Materials were incubated at 37° C. while mixing. At 0, 1, 2 and 4 hoursintervals, samples were collected and analyzed. For endotoxin, nitricoxide (NO=NO₂— plus NO₃—), and hydrogen peroxide (H₂O₂), an additionalseries of experiments were carried out. Endotoxin, NO₂ and NO₃, and H₂O₂were applied in concentrations of 0.777±0.082 EU/ml, 30.18±2.96 μM,238.74±34.86 μM, and 7.76±0.9 nM, respectively. Controls consisted ofspiked FFP only, with no adsorbent present. Materials were incubated at37° C. while mixing (45 deg., 60 cycles/min, Aliquot Mixer, Model 4561,Ames Company, Ames, Iowa). Samples were collected at intervals asdescribed above.

Cytokines/chemokines (TNF-α, IL-1 beta, IL-6, IL-8, IL-10, INF-gamma,MCP-1) were evaluated by the Multi-Analyte Custom ELISArray Kit(CELISA-CMEH0400A, QIAGEN Inc., Valencia, Calif.). This ELISArray Kitswas designed to survey a specific panel of cytokines or chemokinesinvolved in autoimmunity, inflammation, or T-cell biology in cellculture supernatant, serum or plasma. The ELISA was conducted inaccordance with the protocol specified by the manufacturer. The ELISAwas read using Bio-Rad Microplate ELISA reader (Model 3550-UV, Bio-RadLaboratories, Hercules, Calif.) and calculated using Microplate ManagerSoftware Version 2.2 (Bio-Rad Laboratories). NO₂/NO₃=NO concentrationsafter ethyl were established with the Cayman Chemical Nitrate/NitriteAssay Kit (Cat. No. 780001, Ann Arbor, Mich.). Endotoxins (LPS) wereevaluated with QCL-1000 Limulus Amebocyte Lysate Assay Kit (Product No.50-647U, BioWhittaker, Walkersville, Md.). CRP was estimated using thecommercial diagnostic kit from SIGMA Diagnostics (Procedure No. 371-A,St. Louis, Mo.). H₂O₂ was detected by spectrophotometry. The obtainedresults were tabulated, graphically expressed and analyzed.

The results of target molecules clearances are presented in FIGS. 2A-Hwhich depict the effects of mesorporous/microporous synthetic carbon 250or 125 on plasma clearances of: A: TNF-alpha; B: IL-beta; C: IL-6; D:IL-8; E: IL-10; F: MCP-1; G: IFN-gamma; and H: CRP and FIGS. 3A-D whichdepict the effects of mesorporous/microporous synthetic carbon 250 or125 on plasma clearances of: A: nitrite; B: nitrate; C: endotoxin; andD: hydrogen peroxide.

The results demonstrate that synthetic carbons acted non-specifically inremoving EVD target molecules based on their molecular mass. Bothcarbons, 250 and 125, effectively cleared all small inorganic andorganic molecules. Synthetic carbon 250 was more specific toward largerpeptides/proteins (ie, TNF-alpha) and carbon 125 toward smaller ones.

Example Two

Removal of EVD-related Mediators by Anion Exchange Resin From HumanFresh Frozen Plasma.

UNOsphere Q Media (Bio-Rad, Hercules, Calif.), was equilibrated prior totesting, and evaluated for the removal of a range of target moleculesfrom certified human fresh frozen plasma (FFP). Prior to treatment, FFP(Lot No. 9247350, Sera Care Life Sciences, Millford, Mass.) was spikedwith human cytokines (QIAGEN, Inc., Valencia, Calif.) at the followingconcentrations (pg/mL): TNF-α (100-225); IL-1 beta (70-110); IL-8(225-475); IL-10 (85-125); TGF-beta 1 (450 -1,450); IL-6 (80-115);INF-gamma (130-520); IL-12 (85-120); MCP-1 (120-160); MIP-1 alpha(80-120). Materials were incubated at 37° C. while mixing. At 0, 1, 2and 4 hours intervals, samples were collected and analyzed for specificcytokine levels. For endotoxin, NO (NO₂—/NO₃—) and hydrogen peroxide(H₂O₂) an additional series of experiments was carried out. Endotoxin,NO₂, NO₃, and H2O₂ were applied in concentrations of 0.777±0.082 EU/ml,30.18±2.96 μM, 238.74±34.86 μM, and 18.5±3.0, respectively. Controlsconsisted of spiked FFP only, with no adsorbent present. Materials wereincubated at 37° C. while mixing (45 deg, 60 cycles/min, Aliquot Mixer,Model 4561, Ames Company, Ames, Iowa). Samples were collected atintervals as described for cytokines and analyzed.

Cytokines/chemokines (TNF-α, IL-1 beta, IL-8, IL-10, TGF-beta 1, IL-6,INF-gamma, IL-12, MCP-1, MIP-1 alpha) were evaluated by theMulti-Analyte Custom ELISArray Kit (CELISA-CMEH0400A, QIAGEN Inc.,Valencia, Calif.). This ELISArray Kits was designed to survey a specificpanel of cytokines or chemokines involved in autoimmunity, inflammation,or T-cell biology in cell culture supernatant, serum or plasma. TheELISA was conducted in accordance with the protocol specified by themanufacturer. The ELISA was read using Bio-Rad Microplate ELISA reader(Model 3550-UV, Bio-Rad Laboratories, Hercules, Calif.) and calculatedusing Microplate Manager Software Version 2.2 (Bio-Rad Laboratories).NO₂/NO₃=NO concentrations after ethyl were established with the CaymanChemical Nitrate/Nitrite Assay Kit (Cat. No. 780001, Ann Arbor, Mich.).Endotoxins (LPS) were evaluated with QCL-1000 Limulus Amebocyte LysateAssay Kit (Product No. 50-647U, BioWhittaker, Walkersville, Md.). H₂O₂was measured using spectrophometric method.

The results of target molecules clearances are presented in FIG. 3 A, B,C, D, E & F which correspond to clearances of: A: TNF-alpha, IL-1 beta,IL-6; B: IL-8; C: IL-10, MCP-1, MIP-1; D: TGF-beta 1, INF-gamma; E:Nitrite, Nitrate, Hydrogen peroxide; and F: Endotoxin, respectively.

The performed analyses demonstrate that Q Media removed a substantialamount of EVD disease mediators. Q Media had positive effects onpeptide-, protein-based inflammatory mediators and small molecules thatare electronegative in charge at physiologic pH.

Example Three

Removal of EVD-related Mediators by Cation Exchange Resin From HumanFresh Frozen Plasma.

UNOsphere S Media (Bio-Rad, Hercules, Calif.), was equilibrated prior totesting, and evaluated for the removal of a range of target moleculesfrom certified human fresh frozen plasma (FFP). Prior to treatment, FFP(Lot No. 9247350, Sera Care Life Sciences, Millford, Mass.) was spikedwith human cytokines (QIAGEN, Inc., Valencia, Calif.) at theconcentrations provided in TABLE 2 with: TNF-α; IL-1 beta; IL-2; IL-6;IL-10; TGF-beta 1; INF-gamma; and IL-4. Materials were incubated at 37°C. while mixing. At 0, 1, 2 and 4 hours intervals, samples werecollected and analyzed for specific cytokine levels. For endotoxin andNO, an additional series of experiments were carried out. Controlsconsisted of spiked FFP only, with no adsorbent present. Materials wereincubated at 37° C. while mixing (45 deg., 60 cycles/min, Aliquot Mixer,Model 4561, Ames Company, Ames, Iowa). Samples were collected atintervals as described for cytokines and analyzed.

Cytokines/chemokines (TNF-α, IL-1 beta, IL-2, IL-4, IL-6 IL-10, TGF-beta1, INF-gamma) were evaluated by the Multi-Analyte Custom ELISArray Kit(CELISA-CMEH0400A, QIAGEN Inc., Valencia, Calif.). This ELISArray Kitswas designed to survey a specific panel of cytokines or chemokinesinvolved in autoimmunity, inflammation, or T-cell biology in cellculture supernatant, serum or plasma. The ELISA was conducted inaccordance with the protocol specified by the manufacturer. The ELISAwas read using Bio-Rad Microplate ELISA reader (Model 3550-UV, Bio-RadLaboratories, Hercules, Calif.) and calculated using Microplate ManagerSoftware Version 2.2 (Bio-Rad Laboratories). NO concentrations wereestablished with the Cayman Chemical Nitrate/Nitrite Assay Kit (Cat. No.780001, Ann Arbor, Mich.). Endotoxins (LPS) were evaluated with QCL-1000Limulus Amebocyte Lysate Assay Kit (Product No. 50-647U, BioWhittaker,Walkersville, Md.).

The results of target molecules clearances are presented in TABLE 2. Theresults demonstrate that S Media showed the cationic exchangeproperties, targeting molecules that are electropositively charged underthe physiological pH. The cationic inflammatory cytokines such asTGF-beta 1, IL-4 and IFN-gamma were effectively cleared. Thischromatographic resin did not react with albumin, endotoxin and NO.

TABLE 2 sorbing sorbing per gram per gram of de- of de- time changewatered change watered S # analyte (hr) run # 1 (%) S Media run # 2 (%)Media 1 NO3 0 59.65 59.65 (umol) 65.01 1 64.2 (−)7.63 N/A 56.23 5.7348.84 umol umol 4 56.81 4.81 40.56 umol 63.63 (−)6.67 N/A 2 endotoxin 00.243 0.277 (EU/mL) 0.27 EU/mL 1 0.151 37.86 1.31 EU 0.198 28.52 1.13 EU4 0.185 23.87 0.83 EU 0.151 45.49 1.80 EU 3 TGF beta 0 2.45 2.32 1(ng/mL) cationic molecule 2.50 ng/mL 1 0.06 97.55 34.13 ng 0.07 96.9832.13 ng 4 0.05 97.96 34.99 ng 0.06 97.41 32.27 ng 4 IL-1 beta 0 27.7228.5 (pg/mL) neutral molecule 30.5 pg/mL 1 29.1 (−)4.98 N/A 29.95(−)5.09 N/A 4 25.89 6.6 26.13 pg 32.43 (−)13.79 N/A 5 IL-2 0 403.15387.59 (pg/mL) neutral molecule 435.7 pg/mL 1 423.89 (−)10.89 N/A 347.4110.37 573.77 pg 4 434.26 (−)7.72 N/A 383.7 1 55.55 pg 6 IL-4 0 360.99365.11 (pg/mL) cationic molecule 375.07 pg/mL 1 222.48 38.37 1,977.92 pg187.86 48.55 2,531.99 pg 4 151.71 57.97 4,930.74 pg 163.18 55.312,883.56 pg 7 IFN 0 301.11 326.5 gamma (pg/mL) cationic molecule 325.8pg/mL 1 198.06 34.22 1,471.55 pg 164.8 49.53 2,309.08 pg 4 101.4 66.322,851.56 pg 123.51 62.17 2,898.70 pg 8 IL-6 0 308.14 284.46 (pg/mL)anionic molecule 350.72 pg/mL 1 359.79 (−)16.76 N/A 343.26 (−)20.67 N/A4 300.99 2.32 102.10 pg 297.49 (−)4.58 N/A 9 IL-10 0 424.08 480 (pg/mL)anionic molecule 475.1 pg/mL 1 419.26 1.14 68.83 pg 464.96 (−)3.13 N/A 4411.26 3.02 183.07 pg 489.37 (−)1.95 N/A 10 albumin 0 3.4 3.4 (g/dL) 3.5g/dL 1 3.2 5.88 2.86 g 3.1 8.82 4.28 g 4 3 11.76 5.71 g 3 11.76 5.71 g

Example Four

Removal of EVD-related Mediators by Mesorporous/Microporous SyntheticCarbon Beads from Human Whole Blood.

Two formulations of mesoporous/microporous synthetic carbon (125/250 &250/500) were brought to pharmaceutical grade using validatedsanitization and fine particulates removal methods. Thenmesoporous/microporous synthetic carbon beads were used in the agitationtesting protocol identical to that described in Examples 1-3, or packedinto adsorbing devices in sizes representing a 36x scale down from theaverage human extracorporeal circuit (ECMO) model. Prior to testing, thecarbon beads were treated/coated with a solution containing 1% dextranin 09% NaCl, USP (NDC 0409-7419-03, Hospira, Lake Forest, Ill.) and3,000 U HMW heparin (Heparin, Sodium Injection, USP, 1000 U/mL, NDC0641-2440-41 6505-00-153-9740, Elkins-Sinn, Inc., Cherry Hill, N.J.),and later in the agitation experiment combined with 15 mL of spikedfresh human blood or in extracorporeal experiment filled with 76 mL ofspiked fresh human whole blood, warmed to 37° C. Before spiking, humanwhole blood was filtered using 20 μm Pall filter, which was disconnectedduring testing. In the extracorporeal experiment, the back-pressuredetermined the flow rate generated by a peristaltic pump. In bothexperiments, sampling occurred at 0, 1, and 4 hours. In the agitationexperiments tubes containing carbon beads were mixed horizontally, andin the extracorporeal experiments the cartridges containing carbonadsorbents were oriented vertically. In both experiments done induplicates, human whole blood was spiked with inflammatory cytokines(TNF-α, IL-1β, IL-4. IL-6, IL-8, IL-10, IFN-gγ, TGF-β 1), endotoxin, NO,and H₂O₂ thereby, mimicking EVD state.

Cytokines/chemokines (TNF-α, IL-1 β, IL-4, IL-6, IL-8, IL-10, TGF-β 1,IFN-γ) were evaluated by the Multi-Analyte Custom ELISArray Kit(CELISA-CMEH0400A, QIAGEN Inc., Valencia, Calif.). This ELISArray Kitswas designed to survey a specific panel of cytokines or chemokinesinvolved in autoimmunity, inflammation, or T-cell biology in cellculture supernatant, serum or plasma. The ELISA was conducted inaccordance with the protocol specified by the manufacturer. The ELISAwas read using Bio-Rad Microplate ELISA reader (Model 3550-UV, Bio-RadLaboratories, Hercules, Calif.) and calculated using Microplate ManagerSoftware Version 2.2 (Bio-Rad Laboratories). NO₂/NO₃=NO concentrationsafter ethyl were established with the Cayman Chemical Nitrate/NitriteAssay Kit (Cat. No. 780001, Ann Arbor, Mich.). Endotoxins (LPS) wereevaluated with QCL-1000 Limulus Amebocyte Lysate Assay Kit (Product No.50-647U, BioWhittaker, Walkersville, Md.). CRP was estimated using thecommercial diagnostic kit from SIGMA Diagnostics (Procedure No. 371-A,St. Louis, Mo.). Hydrogen peroxide was assayed spectrophotometrically.The results of target molecule clearances are presented in TABLES 3-14for extracorporeal testing and TABLES 3A-14A for agitation testing.

The results demonstrate that mesoporous/microporous synthetic carbonbeads were effective in removal of EVD mediators responsible for SIRS,immune system suppression, hypotension and MOF. Synthetic carbon beadsin experimental conditions investigated, extracorporeal and agitation,showed similar cleansing effectiveness toward EVD mediators.

TABLE 3 INTERFEREON GAMMA RUN 1 RUN 2 pg/ pg/ pg/mL 76 mL pg/mL 76 mLTime whole whole pg per g Time whole whole pg per g (hr) blood bloodsorbent (hr) blood blood sorbent 0 87.31 6,635 — 0 — — — 1 25.29 1,922589.1 1 — — — 4 27.69 2,105 566.2 4 — — —

TABLE 3A INTERFEREON GAMMA pg/15 pg/mL mL Time whole whole pg per g (hr)blood blood sorbent RUN 1 0 86.70 1,300.5 — 1 28.90 433.5 548.7 4 28.9 433.5 548.7 RUN 2 0 — — — 1 — — — 4 — — —

TABLE 4 C-reactive protein mg/dL Time whole % (hr) blood decrease RUN 10 2.01  — 1 0.157 92.2 4  0.377- 81.2 RUN 2 0 — — — 1 — — — 4 — — —

TABLE 4A C-reactive protein mg/dL Time whole % (hr) blood decrease RUN 10 2.29  — 1 0.472 79.4  4 0.251 89.03 RUN 2 0 — — — 1 — — — 4 — — —

TABLE 5 ENDOTOXIN EU/76 EU/mL mL Time whole whole EU per g (hr) bloodblood sorbent RUN 1 0 0.747 58.82 — 1 0.205 15.58 5.405 4 0.178 13.535.661 RUN 2 0 0.755 57.38 — 1 0.190 14.47 5.364 4 0.175 13.32 5.507

TABLE 5A ENDOTOXIN EU/15 EU/mL mL Time whole whole EU per g (hr) bloodblood sorbent RUN 1 0 0.831 12.46 — 1 0.262 3.926 5.401 4 0.102 1.5376.913 RUN 2 0 0.757 11.359 — 1 0.228 3.426 4.989 4 0.173 3.595 5.547

TABLE 6 TGF-β 1 pg/76 pg/mL mL Time whole whole pg per g (hr) bloodblood sorbent RUN 1 0 1,303.5 99,066 — 1 1,149.3 87,346 1,465 4 778.9559,200 4,983 RUN 2 0 1,291 98,116 — 1 1,081 82,156 1,995 4 738.1 56,0955,252

TABLE 6A TGF-β 1 pg/15 pg/mL mL Time whole whole pg per g (hr) bloodblood sorbent RUN 1 0 1,249 18,735 — 1 855 12.825 3,740 4 783 11,7454,424 RUN 2 0 1,303 19,545 — 1 950.2 14,253 3,349 4 768.4 11,520 5,063

TABLE 7 TNF-α pg/76 pg/mL mL Time whole whole pg per g (hr) blood bloodsorbent RUN 1 0 1,898.9 144,323 — 1 1,506.2 114,469  3,731.7 4 825.4 62,735 10,198.7 RUN 2 0 — — — 1 — — — 4 — — —

TABLE 7A TNF-α pg/15 pg/mL mL Time whole whole pg per g (hr) blood bloodsorbent RUN 1 0 2,007.1 30,106.5 — 1 1,614.3 24,214.5 3,729.1 4 681.510,222.8 12,584.6 RUN 2 0 — — — 1 — — — 4 — — —

TABLE 8 IL-4 pg/76 pg/mL mL Time whole whole pg per g (hr) blood bloodsorbent RUN 1 0 54.29 4,125.8 — 1 48.99 3,723.8 50.25 4 38.69 2,940.7148.1  RUN 2 0 40.83 3,103.16 — 1 37.54 2,853.04 31.26 4 32.92 2,501.8475.16

TABLE 8A IL-4 pg/15 pg/mL mL Time whole whole pg per g (hr) blood bloodsorbent RUN 1 0 71.03 1,065.5 — 1 66.41 996.23 43.84 4 62.37 935.5982.22 RUN 2 0 76.23 1,143.4 — 1 76.23 1,143.4 0 4 62.37 935.59 131.52

TABLE 9 IL-6 pg/76 pg/mL mL Time whole whole pg per g (hr) blood bloodsorbent RUN 1 0 5,467.8 415,550 — 1 2,835.5 215,500 25,006 4 1,473.9112,020 37,941 RUN 2 0 4,619.4 351,080 — 1 3,308.1 251,420 12,450 41,551.4 117,910 29,150

TABLE 9A IL-6 pg/15 pg/mL mL Time whole whole pg per g (hr) blood bloodsorbent RUN 1 0 4,958.5 74,378 — 1 1,944.6 29,169 28,610 4 999.4 14,99137,590 RUN 2 0 4,673 70,105 — 1 1,696 25,450 28,260 4 859 12,899 36,210

TABLE 10 IL-8 pg/76 pg/mL mL Time whole whole pg per g (hr) blood bloodsorbent RUN 1 0 2,139 162,576 — 1 1,816 138,036 3,067.5 4 1,388 105,5407,129.5 RUN 2 0 2,366 179,845 — 1 1,728.1 131,333 6,064 4 1,367.8103,953 9,486

TABLE 10A IL-8 pg/15 pg/mL mL Time whole whole pg per g (hr) blood bloodsorbent RUN 1 0 1,955.3 29,329 — 1 1,204.8 18,072  7,125 4 837.1 12,55610,615 RUN 2 0 2,054 30,809 — 1 1,432 21,481  5,904 4 734 11,010 12,531

TABLE 11 IL-10 pg/76 pg/mL mL Time whole whole pg per g (hr) blood bloodsorbent RUN 1 0 3,004 228,304 — 1 997 75,772 19,066 4 343.8 26,12925,272 RUN 2 0 2,381.8 181,017 — 1 675.7 51,353 16,208 4 169.5 12.88221,017

TABLE 11A IL-10 pg/15 pg/mL mL Time whole whole pg per g (hr) bloodblood sorbent RUN 1 0 3,214 48,210 — 1 1,767.6 26,514 13,732 4 723.210,848 23,647 RUN 2 0 3,240 48,600 — 1 1,751.1 26,266 14,135 4 621.2 9,318 24,862

TABLE 12 IL-1β pg/76 pg/mL mL Time whole whole pg per g (hr) blood bloodsorbent RUN 1 0 154.52 11,743.5 — 1 84.98 6,458.5 660 4 0 0 1,467.9 RUN2 0 150.65 11,449 — 1 88.84 6,752 587.1 4 0 0 1,431.1

TABLE 12A IL-1 β pg/15 pg/mL mL Time whole whole pg per g (hr) bloodblood sorbent RUN 1 0 118.98 1,784.7 — 1 42.58 638.71 725 4 7.72 115.81,056.2 RUN 2 0 122.06 1,831.02 — 1 50.75 761.20 677.1 4 4.63 69.531,114.8

TABLE 13 Nitric Oxide (NO = NO₂ + NO₃) uM Time whole % (hr) blooddecrease RUN 1 0 255.4   — — 1 0.501 95.0 — 4  0.000- 100   — RUN 2 0 —— — 1 — — — 4 — — —

TABLE 13A Nitric Oxide (NO = NO₂ + NO₃) uM Time whole % (hr) blooddecrease RUN 1 0 252.3 — — 1 100.9 60.0 — 4  35.7 85.8 — RUN 2 0 — — — 1— — 4 — — —

TABLE 14 Hydrogen Peroxide (H₂O₂) nM Time whole % (hr) blood decreaseRUN 1 0 235.7  — — 1 85.8 63.6 — 4 27.9 88.2 — RUN 2 0 — — — 1 — — — 4 —— —

TABLE 14A Hydrogen Peroxide (H₂O₂) nM Time whole % (hr) blood decreaseRUN 1 0 9.70  — 1 0.157 92.2 4  0.000- 100   RUN 2 0 — — — 1 — — — 4 — ——

Example Five

Effect of Sanitization of Mesorporus/Microporous Synthetic Carbon Beads(125 & 250), Q Media and S Media on Their Sorbing Capacity TowardEVD-related Mediators and Physiologic Parameters.

De-watered mesoporous/microporous Synthetic Carbon Beads 125 and 250, QMedia and S Media (in weight proportions: 50%. 20%. 20%, 10%,respectively) in nonsterile (industrial grade) and sterile(pharmaceutical grade) forms, were packed in a Bio-Rad chromatographycolumn (ID 65 mm×17 mm) and tested for the removal of the targetmolecules in the EVD condition. After packing, the chromatographicmaterial was primed with 1% LMW Dextran 40 in 0.9% NaCl, USP (NDC0409-7419-03, Hospira). Then the column was connected with theextracorporeal circuit filled with spiked FFP, as described in Example4. FFP extracorporeal cleansing was conducted for 4 hours. Samples werecollected at 0, 1 and 4 hrs and subjected for analyses, as described inprevious Examples. Additionally, physiologic parameters were establishedusing Abaxis Piccolo Xpress Chemistry Analyzer and IRMA TruPoint BloodAnalysis System. Table 15 provides metrics regarding the adsorbentmaterial's clearance for the indicated compound (parameter) beforesanitization (Industrial Grade) while Table 16 provides metricsregarding the adsorbent material's clearance for the indicated compoundafter sanitization (Pharmaceutical Grade).

The results presented in Tables 15 and 16 demonstrate adsorbentsformulations, which contained synthetic carbons 250 and 125, Q Media andS Media ,not subjected for sanitization, very effectively removedmediators of EVD and the effect on physiological mediators was minimal.It was further noted that sanitization of adsorbents (synthetic carbons250 and 125, Q Media and S Media), had almost no effect on theirsorbing/binding potency toward EVD mediators.

TABLE 15 % % PARAMETER 0 HR 1 HR CHANGE 4 HR CHANGE TNF alpha 759.59282.81 −62.8 183.37 −75.8 (pg/mL) IL-1 beta 156.59 90.91 −41.9 86.01−45.1 (pg/mL) IL-6 (pg/mL) 325.20 92.63 −71.5 61.32 −81.1 IL-8 (pg/mL)881.76 217.43 −75.3 101.22 −88.5 IL-10 (pg/mL) 934.45 393.62 −57.9218.72 −76.6 TGF beta 1 1393.75 109.25 −92.2 45.93 −96.7 (pg/mL)endotoxin 1.036 0.131 −87.3 0.149 −85.6 (EU/mL) C3a des Arg 3192.21562.3 −51.0 668.9 −79.0 (ng/mL) nitric 95.39 48.70 −48.9 58.54 −38.6oxide (uM) ALB (g/dL) 4.4 4.1 −6.8 3.9 −11.4 ALP (U/L) 41 35 −14.6 37−9.7 ALT (U/L) 15 8 −46.7 6 −60.0 AMY (U/L) 84 54 −35.7 53 −36.9 TBIL(mg/dL) 0.7 0.7 0 0.6 −14.3 BUN (mg/dL) 12 10 −16.7 11 −8.3 Ca (mg/dL)6.5 4.9 −24.6 4.9 −24.6 PHOS (mg/dL) 9.7 9.0 −7.2 8.9 −8.2 CRE (mg/dL)0.9 0.2 −77.8 0.3 −66.7 GLU (mg/dL) 341 268 −21.4 265 −22.3 Na+ (mM) 157150 −4.4 146 −7.0 K+ (mM) 3.7 3.4 −8.1 3.3 −10.8 TP (g/dL) 6.9 6.4 −7.26.0 −13.0 GLOB (g/dL) 2.5 2.3 −8.0 2.1 −16.0 pH 7.579 7.490 1.2

TABLE 16 % % PARAMETER 0 HR 1 HR CHANGE 4 HR CHANGE TNF alpha 787.15304.24 −61.3 294.16 −62.6 (pg/mL) IL-1 beta 162.51 80.28 −50.6 103.52−36.3 (pg/mL) IL-6 (pg/mL) 437.36 138.70 −68.3 20.38 −95.3 IL-8 (pg/mL)952.76 225.82 −76.3 104.81 −89.0 IL-10 (pg/mL) 920.25 432.49 −53.0205.66 −77.6 TGF beta 1 1402.45 163.66 −88.3 157.58 −88.8 (pg/mL)endotoxin 1.015 0.151 −85.1 0.123 −87.9 (EU/mL) C3a des Arg 3322.61808.5 −45.6 330.2 −90.1 (ng/mL) nitric 95.20 57.32 −39.8 61.39 −35.5oxide (uM) ALB (g/dL) 4.4 4.1 −6.8 4.0 −9.0 ALP (U/L) 40 37 −7.5 31−22.5 ALT (U/L) 9 16 77.8 12 −33.3 AMY (U/L) 62 55 −11.3 53 −14.5 TBIL(mg/dL) 0.6 0.6 0 0.6 0 BUN (mg/dL) 12 11 −8.3 12 0 Ca (mg/dL) 6.6 5.0−24.2 5.0 −24.2 PHOS (mg/dL) 9.6 9.2 −4.2 8.8 −8.3 CRE (mg/dL) 1.0 0.4−60.0 0.2 −80.0 GLU (mg/dL) 347 264 −76.0 266 −23.3 Na+ (mM) 156 150−3.8 148 −5.1 K+ (mM) 3.9 3.4 −12.8 3.4 −12.8 TP (g/dL) 7.1 6.4 −9.8 6.4−9.8 GLOB (g/dL) 2.7 2.3 −14.8 2.5 −7.4 pH 7.586 7.549 0.5

Example Six

Impact of Priming/Coating of Mesoporous/Microporous Synthetic Carbonswith LMW Dextran on Their Sorbing Capacity Toward Small and LargeMolecules.

Mesoporous/micrporous synthetic carbon beads were primed/coated withdextran 40 in concentrations of: 0, 1, 2.5, 5, 7.5 and 10%, thenutilized for extracorporeal removal of small molecules (radiocontrastOptiray 350) and large molecules (human albumin). The experiments weredone as described in Example 4. Human albumin levels were established onAbaxis Piccolo Xpress Blood Chemistry Analyzer and radiocontrast with UVspectrophotometry. The results of coating mesoporous/microporoussynthetic carbon beads with LMW dextran 40 on large molecule clearances(human albumin) are depicted in FIG. 4A while the results of coatingmesoporous/microporous synthetic carbon beads with LMW dextran 40 onsmall molecule clearances (radiocontrast Optiray 350) is depicted inFIG. 4B.

The results demonstrate the observed sorbing modulation effect towardsmall and large molecules was time and concentration dependent. Dextranin higher concentrations (7.5-10%) obstructed the removal of bothmolecules. Dextran in lower concentrations (2.5-5%) showed a moreenhanced removal, while 1% dextran had no effect. The results suggestdextran in highly concentrated form can be used as a method to restricthigher molecular weight compounds and permit sorption of smallmolecules. Without wishing to be limited by theory, the proposedmechanism of observed dextran-controlled sorbing activity can beexplained by nonspecific incorporation of different numbers of 40 kDadextran molecules into synthetic carbon pores, and their removal overtime in exchange for target molecules. Besides, LMW dextran can serve asa lubricant during storage/shipment of devices containing syntheticcarbon beads, by preventing formation of new fine particulates.

Example Seven

Animal Safety Study of Adsorbing Devices Containing Synthetic Carbon250, Synthetic Carbon 125, Q Media and S Media.

This study, which received clearance from the TTUHSC IACUC (Protocol No.13003), was designed to investigate the safety of the treatment withadsorption devices in a dog model. Experimental animals underwent asix-hour extracorporeal treatment with device containing 4 adsorbents:synthetic carbon 250, synthetic carbon 125, Q Media and S Media used inde-watered weight proportions: 50%. 20%. 20%, 10%, respectively. Theadsorption devices were sterile, pyrogen free and highlyhemo-biocompatible.

The animal study was designed to obtain information about clinicalperformance of the adsorption devices in healthy anesthetized dogs. Thisstudy determined the effect of the ImMutriX Adsorption Devices over 6hours on: (i) hemodynamics; (ii) hematologic parameters; (iii)analytical blood chemistry parameters; and (iv) anatomo-histopathology.Data were collected at:—baseline after induction of anesthesia/catheterplacement & prior to adsorption device treatment. Baseline laboratoryconsisted of: AST, ALT, LD, ALP, Albumin, Globulins, Ammonia, lacticacid, total protein, bilirubin, pH, pO₂, pCO₂, Na⁺, K⁺, Cl⁻, iCa⁺⁺, tCa,TCO₂, HCO₃, BEb, BEecf, tCa, Mg, BUN and Creatinine, Glucose, completeblood count (Hg, Hct, RBC, WBC, differential, and platelets),coagulation panel: aPTT, PT, INR, D-Dimer and fibrinogen, hemodynamicmonitoring (Vital signs Q15 min/Hemodynamics Q30 min), vital signs(blood pressure —M, S/D, oxygen saturation & temperature), hemodynamics(PAP, PCWP, CVP, CO, PVR & SVR), urinary output. During the treatmentat: 30 min, 60 min, 120 min, 180 min, 240 min, 300 min, and 360 min(some parameters, including hemodynamics were also measured at 15minutes, 90 min and 150 min), AST, ALT, LD, ALP, Albumin, Globulins,Ammonia, lactic acid, total protein, bilirubin, pH, pO₂, pCO₂, Na⁺, K⁺,Cl⁻, iCa⁺⁺, tCa, TCO₂, HCO₃, BEb, BEecf, tCa, Mg, BUN and Creatinine,Glucose, complete blood count (Hg, Hct, RBC, WBC, differential, andplatelets), coagulation panel: aPTT, PT, INR, D-Dimer and fibrinogen,hemodynamic monitoring (Vital signs Q15 min/Hemodynamics Q30 min), vitalsigns (blood pressure—M, S/D, oxygen saturation &temperature),hemodynamics (PAP, PCWP, CVP, CO, PVR & SVR), urinaryoutput. Necropsy and histopathological evaluation (H&E) of lung, liver,kidney, heart, and GI tract were also performed.

Tables 17-24 provide summaries of the data for the animal study. Therecording of all hemodynamic parameters allowed a comprehensiveevaluation of the hemodynamic effects of the adsorbing devices. Acareful analysis of these parameters illustrates that the devices didnot cause any significant changes in MAP, CVP, PCWP, PAP, SVR and PVR.Overall, the hemodynamic data leads to the conclusion that six hourextracorporeal treatment with the adsorbing devices was well toleratedby dogs.

TABLE 17 Baseline 15 30 60 90 120 150 180 210 240 270 300 330 360 (3-1)(3-1-1) (3-2) (3-3) (3-3-1) (3-4) (3-4-1) (3-5) (3-5-1) (3-6) (3-6-1)(3-7) (3-7-1) (3-8) HR 98 96 93 93 92 90 90 89 90 87 89 90 77 82 (b/min)BP - 94/52 110/54 110/58 118/65 115/60 105/53 103/53 105/46 105/53109/53 95/48 102/43 107/53 118/58 S/D (mmHg) BP - 65 90 75 85 78 64 6864 70 71 61 61 72 79 M (mmHg) CVP 5 4 2 5 10 10 10 10 8 10 13 13 13 13(mmHg) PA - 34/18 27/8 24/8 22/7  30/18  24/14  24/14  29/17  24/14 29/18 25/16  31/20  32/22  28/18 S/D (mmHg) PA - 23.3 14.3 13.3 12 2217.3 17.3 21 17.3 21.7 19 23.7 25.3 21.3 M (mmHg) PCWP — — — — — — — — —— — — — — (mmHg) Not For PVR: recorded arbitrary: 10 CO 4.1 5.8 4.1 4.04.0 3.9 4.3 4.3 3.8 3.9 3.7 3.9 4.5 4.0 (L/min) PVR 259 59.3 64.4 40 240149.7 135.8 204.6 153.7 240 194.6 266.7 272 226 (dyne s cm{circumflexover ( )}−5) SVR 1169 1171 1364 1598 1358 1106 1077 1096 1303 1250 1144983 1047 1318 (dyne s cm{circumflex over ( )}−5) Urine 60 0 20 15 23 1015 10 15 17 7 23 10 34 Output (mL)

TABLE 18 Baseline 15 30 60 90 120 150 180 210 240 270 300 330 360 (3-1)(3-1-1) (3-2) (3-3) (3-3-1) (3-4) (3-4-1) (3-5) (3-5-1) (3-6) (3-6-1)(3-7) (3-7-1) (3-8) pH 7.405 7.306 7.322 7.318 — 7.352 — 7.405 — 7.392 —7.372 — 7.380 (U) pCO2 31.3 39.2 47.7 47.0 — 40.1 — 37.0 — 41.2 — 42.8 —42.9 (mmHg) pO2 321.6 309.9 380.0 353.6 — 318.9 — 168.8 — 371.8 — 381.7— 388.5 (mmHg) Na+ 148.8 149.4 150.2 150.7 — 150.4 — 147.0 — 147.2 —146.7 — 146.6 (mM) K+ 2.93 2.68 2.84 2.78 — 2.69 — 3.44 — 3.20 — 3.02 —2.92 (mM) iCa++ 1.47 1.16 1.27 1.31 — 1.22 — 1.23 — 1.33 — 1.36 — 1.30(mM) HCO3− 19.4 19.4 24.4 23.8 — 22.0 — 22.9 — 24.7 — 24.6 — 25.1 (mM)TCO2 20.3 20.6 25.9 25.3 — 23.2 — 24.0 — 26.0 — 25.9 — 26.4 (mM) BEb−3.6 −5.8 −1.5 −2.0 — −2.7 — −0.8 — 0.4 — −0.2 — 0.4 (mM) BEecf −5.6−7.2 −1.9 −2.5 — −3.8 — −2.0 — −0.4 — −0.9 — −0.3 (mM) O2Sat 99.7 99.799.8 99.7 — 99.7 — 99.1 — 99.8 — 99.8 — 99.8 (%)

TABLE 19 Baseline 15 30 60 90 120 150 180 210 240 270 300 330 360 (3-1)(3-1-1) (3-2) (3-3) (3-3-1) (3-4) (3-4-1) (3-5) (3-5-1) (3-6) (3-6-1)(3-7) (3-7-1) (3-8) THb 9.8 — 12.1 12.0 — 9.8 — 10.3 — 9.9 — 10.4 — 8.9(g/dL) OxyHb 90.4 — 90.2 90.4 — 90.9 — 89.9 — 90.4 — 90.5 — 89.6 (%)COHb 9.9 — 10.0 9.8 — 10.1 — 9.1 — 9.7 — 10.0 — 9.7 (%) MetHb 0.5 — 0.50.5 — −0.3 — 0.9 — 0.5 — 0.4 — 0.9 (%) RHb −0.7 — −0.7 −0.7 — −0.7 — 0.1— −0.6 — −0.9 — −0.2 (Reduced Hb - %) O2 12.3 — 15.2 15.0 — 12.4 — 12.9— 12.4 — 13.1 — 11.1 Content (mL/dL) O2 12.2 — 15.1 15.0 — 12.3 — 12.9 —12.4 — 13.0 — 11.1 Capacity (mL/dL)

TABLE 20 Baseline 15 30 60 90 120 150 180 210 240 270 300 330 360 (3-1)(3-1-1) (3-2) (3-3) (3-3-1) (3-4) (3-4-1) (3-5) (3-5-1) (3-6) (3-6-1)(3-7) (3-7-1) (3-8) Sodium 141 — 137 137 — 136 — 138 — 136 — 135 — 137mmol/L (128-145) Potassium 3.0 — 2.9 3.0 — 2.9 — 3.4 — 3.3 — 3.1 — 3.2mmol/L (3.6-5.1) Chloride 110 — 111 112 — 114 — 111 — 112 — 110 — 109mmol/L (90-108) Total CO2 20 — 25 25 — 23 — 26 — 26 — 26 — 27 mmol/L(18-33) Creatinine 0.6 — 0.6 0.7 — 0.4 — 0.3 — 0.5 — 0.2 — 0.4 mg/dL(0.6-1.2) BUN 14 — 14 14 — 12 — 11 — 12 — 11 — 11 mg/dL (7-22) Glucose150 — 156 166 — 146 — 168 — 167 — 175 — 164 mg/dL (73-118) Total 10.2 —7.5 8.2 — 7.1 — 7.8 — 8.1 8.0 8.1 Calcium mg/dL (8.0-10.3) Magnesium 1.6— 1.4 1.5 — 1.3 — 1.6 — 1.6 1.6 1.7 mg/dL (1.6-2.3) Lactate 195 — 96 128— 97 — 69 — 54 73 76 Dehydro- genase U/L (99-192)

TABLE 21 Baseline 15 30 60 90 120 150 180 210 240 270 300 330 360 (3-1)(3-1-1) (3-2) (3-3) (3-3-1) (3-4) (3-4-1) (3-5) (3-5-1) (3-6) (3-6-1)(3-7) (3-7-1) (3-8) Albumin 1.6 — 1.6 1.6 — 1.2 1.2 1.2 — 1.3 — 1.4 —1.3 g/dL (2.5-4.4) ALP 35 — 36 36 — 39 39 40 — 48 — 46 — 53 U/L (20-150)ALT 12 — 14 10 — 11 11 13 — 14 — 15 — 14 U/L (10-118) Amylase 367 — 381393 — 338 334 369 — 401 — 417 — 402 U/L (200-1200) Total 0.3 — 0.3 0.3 —0.2 0.3 0.3 — 0.3 — 0.3 — 0.3 Bilirubin mg/dL (0.1-0.6) BUN 13 — 14 13 —11 11 12 — 12 — 11 — 10 mg/dL (7-25) Total 7.1 — 7.9 8.0 — 6.9 7.0 7.3 —8.0 — 7.9 — 7.8 Calcium mg/dL (8.6-11.8) Phosphorus 5.2 — 6.4 6.1 — 5.25.7 6.0 — 5.7 — 5.3 — 5.1 mg/dL (2.9-6.6) Creatinine 1.0 — 0.9 1.2 — 0.70.6 0.4 — 0.6 — 0.5 — 0.5 mg/dL (0.3-1.4) Glucose 135 — 151 161 — 144142 165 — 165 — 172 — 163 mg/dL (60-110) Sodium 139 — 139 139 — 139 139138 — 138 — 138 — 139 mmol/L (138-160) Potassium 2.3 — 2.5 2.5 — 2.3 2.23.2 — 3.0 — 3.0 — 2.6 mmol/L (3.7-5.8) Total 3.1 — 3.1 3.1 — 2.6 2.6 2.7— 2.7 — 2.7 — 2.7 Protein g/dL (5.4-8.2) Globulin 1.5 — 1.5 1.5 — — 1.51.5 — 1.5 — 0.0 — 0.0 g/dL (2.3-5.2)

TABLE 22 Baseline 15 30 60 90 120 150 180 210 240 270 300 330 360 (3-1)(3-1-1) (3-2) (3-3) (3-3-1) (3-4) (3-4-1) (3-5) (3-5-1) (3-6) (3-6-1)(3-7) (3-7-1) (3-8) PT 8.4 — 9.8 9.4 — 11.9 — 13.7 — 13.1 — 12.7 — 15.0sec (9.3-12.1) International 0.75 — 0.88 0.84 — 1.07 — 1.24 — 1.19 —1.15 — 1.36 Normalized Ratio (INR) PTT 178.0 >400 216.4 101.7 >400 217.0237.6 187.3 145.6 197.4 156.6 >400 311.0 211.5 sec (26.1-37.9)Fibrinogen 56.0 — 78.0 57.0 — 54.0 — 51.0 — 56.0 — 55.0 — 52.0 mg/dL(197-406) D-Dimer 165 — <150 <150 — <150 — <150 — <150 — <150 — <150ng/mL (0-243)

TABLE 23 Baseline 15 30 60 90 120 150 180 210 240 270 300 330 360 (3-1)(3-1-1) (3-2) (3-3) (3-3-1) (3-4) (3-4-1) (3-5) (3-5-1) (3-6) (3-6-1)(3-7) (3-7-1) (3-8) Lactic 3.2 — 2.5 2.5 — 1.3 — 1.1 — — — 1.1 — 1.3Acid mmol/L (0.5-2.2) Ammonia 21 — 24 27 — 11 — 14 — — — 13 — 27 mcmol/L(56-92)

TABLE 24 Baseline 15 30 60 90 120 150 180 210 240 270 300 330 360 (3-1)(3-1-1) (3-2) (3-3) (3-3-1) (3-4) (3-4-1) (3-5) (3-5-1) (3-6) (3-6-1)(3-7) (3-7-1) (3-8) WBC 3.14 — 3.75 4.22 — 4.04 — 4.97 — 5.88 — 5.82 —6.04 (10{circumflex over ( )}9/L) LYMPH 0.89 — 0.90 1.22 — 0.87 — 0.92 —1.29 — 1.02 — 1.46 (10{circumflex over ( )}9/L) MONO 0.18 — 0.15 0.22 —0.26 — 0.27 — 0.46 — 0.41 — 0.42 (10{circumflex over ( )}9/L) NEUT 2.06— 2.69 2.79 — 2.91 — 3.78 — 4.13 — 4.39 — 4.16 (10{circumflex over( )}9/L) EOS 0.01 — 0.01 0.00 — 0.00 — 0.00 — 0.00 — 0.00 — 0.00(10{circumflex over ( )}9/L) BASO 0.00 — 0.00 0.00 — 0.00 — 0.00 — 0.00— 0.00 — 0.00 (10{circumflex over ( )}9/L) LYMPH 28.3 — 24.0 28.8 — 21.6— 18.4 — 22.0 — 17.5 — 24.2 (%) MONO 5.7 — 4.0 5.1 — 6.4 — 5.4 — 7.8 —7.0 — 6.9 (%) NEUT 65.8 — 71.8 66.0 — 72.0 — 76.1 — 70.2 — 75.4 — 69.8(%) EOS 0.2 — 0.1 0.1 — 0.1 — 0.0 — 0.0 — 0.0 — 0.1 (%) BASO 0.0 — 0.00.0 — 0.0 — 0.0 — 0.0 — 0.0 — 0.0 (%) RBC 4.12 — 4.89 4.92 — 4.06 — 4.17— 3.96 — 4.29 — 3.63 (10{circumflex over ( )}12/L) Hb 9.7 — 11.9 11.6 —9.5 — 9.8 — 9.6 — 10.3 — 8.5 (g/dL) HCT 28.17 — 33.58 34.04 — 28.13 —28.65 — 26.97 — 29.73 — 24.84 (%) MCV 68 — 69 69 — 69 — 69 — 68 — 69 —68 (u) MCH 23.4 — 24.3 23.5 — 23.3 — 23.5 — 24.3 — 24.1 — 23.5 (pg) MCHC34.3 — 35.4 33.9 — 33.7 — 34.2 — 35.6 — 34.8 — 34.3 (g/dL) RDWc 14.8 —15.0 15.2 — 14.6 — 15.0 — 14.6 — 14.8 — 15.0 (%) PLT 113 — 152 149 — 116— 127 — 121 — 120 — 102 (10{circumflex over ( )}9/L) PCT 0.12 — 0.170.16 — 0.13 — 0.13 — 0.13 — 0.14 — 0.11 (%) MPV 10.3 — 11.0 10.7 — 11.1— 10.5 — 11.1 — 11.4 — 10.8 (u) PDWc 36.9 — 39.0 39.2 — 38.9 — 38.6 —38.5 — 39.8 — 38.8 (%)

The following are enumerated embodiments are provided as non-limitingexamples:

A first embodiment which is a method comprising obtaining a bodily fluidfrom a subject having a level of disease mediators (y); contacting thebodily fluid with an adsorbent material comprising an synthetic carbonparticle (SCP) to produce a first filtrate; contacting the firstfiltrate with an adsorbent material comprising the SCP and an anionexchange resin where the weight ratio of SCP to anion exchange resin isfrom about 0.1:100 to 100:0.1 to produce a second filtrate; contactingthe second filtrate with an adsorbent material comprising the SCP and acation exchange resin where the weight ratio of SCP to cation exchangeresin is from about 0.1:100 to 100:0.1 produce a third filtrate; andadministering the third filtrate to the subject.

A second embodiment which is the method of the first embodiment whereinthe subject is diagnosed with or suspected of having viruses associatedwith immunosuppressive events.

A third embodiment which is the method of any of the first throughsecond embodiments wherein the viruses associated with immunosuppressiveevents is a member of a viral family selected from the group consistingof Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae, Coronavirinae,Bunyaviridae, and Orthomyxoviridae.

A fourth embodiment which is the method of any of the second throughthird embodiments wherein the virus comprises Hantavirus,MERS-coronavirus (MERS-CoV), Influenza A virus subtype H5N1, Influenza A(H1N1) virus, Ebola Virus, Marburg virus, or combinations thereof.

A fifth embodiment which is the method of any of the first throughfourth embodiments further comprising sanitizing the SCP, the anionexchange resin, and the cation exchange resin prior to contacting withthe bodily fluids.

A sixth embodiment which is the method of any of the first through fifthembodiments comprising contacting the SCP, the anion exchange resin, andthe cation exchange resin with a compatibilizer prior to contacting theSCP, the anion exchange resin, and the cation exchange resin with thebodily fluids.

A seventh embodiment which is the method of the sixth embodiment whereinthe compatibilizer comprises a polysaccharide, a glucan, albumin,mannitol, a starch, or combinations thereof.

An eighth embodiment which is the method of any of the sixth throughseventh embodiments wherein the compatibilizer comprises dextran.

A ninth embodiment which is the method of the seventh embodiment whereinthe dextran has an average molecular weight of from about 1 kDa to about500 kDa.

A tenth embodiment which is the method of the seventh embodiment whereinthe compatibilizer comprises hydroxyethyl starch.

An eleventh embodiment which is the method of the seventh embodimentwherein the compatibilizer comprises albumin and mannitol.

A twelfth embodiment which is the method of any of the first througheleventh embodiments wherein a level of a disease mediator in the thirdfiltrate is reduced by about 100% when compared to the level of diseasemediators (y).

A thirteenth embodiment which is the method of the twelfth embodimentwherein the disease mediators are selected from the group consisting ofIL-18, IFN-γ, TNF-α, IL-1β, IL-6, IL-10, MCP-1, MCSF, MIP-1 α, NO, C3a,C5a, histamine, and combinations thereof.

A fourteenth embodiment which is an extracorporeal system comprising atleast three adsorbent materials, an access disconnection detector, and acomputer system.

A fifteenth embodiment which is the extracorporeal system of thefourteenth embodiment wherein the adsorbent materials comprise ansynthetic carbon particle, a mixture of an synthetic carbon particle andan anion exchange resin, and a mixture of a synthetic carbon particles(SCP) and a cation exchange resin.

A sixteenth embodiment which is the extracorporeal system of thefourteenth through fifteenth embodiments wherein the SCP and the anionexchange resin are present in a ratio of 1 wt. % SCP to 99 wt. % anionexchange resin.

A seventeenth embodiment which is the extracorporeal system of any ofthe fourteenth through sixteenth embodiments wherein the SCP and thecation exchange resin are present in a ratio of 1 wt. % SCP to 99 wt. %cation exchange resin.

An eighteenth embodiment which is the extracorporeal system of any ofthe fourteenth through seventeenth embodiments wherein the at leastthree adsorbent materials have at least a portion of their surfacecoated with a compatibilizer.

A nineteenth embodiment which is the extracorporeal system of theeighteenth embodiment wherein the compatibilizer comprises a glucan.

A twentieth embodiment which is the extracorporeal system of any of theeighteenth through nineteenth embodiments wherein the compatibilizercomprises dextran.

A twenty-first embodiment which is the extracorporeal system of FIG. 1.

While embodiments of the present disclosure have been shown anddescribed, modifications thereof can be made by one skilled in the artwithout departing from the spirit and teachings of the disclosure. Theembodiments described herein are exemplary only, and are not intended tobe limiting. Many variations and modifications of the disclosure arepossible and are within the scope of the invention. Use of the term“optionally” with respect to any element of a claim is intended to meanthat the subject element is required, or alternatively, is not required.Both alternatives are intended to be within the scope of the claim. Useof broader terms such as comprises, includes, having, etc. should beunderstood to provide support for narrower terms such as consisting of,consisting essentially of, comprised substantially of, etc.

Accordingly, the scope of protection is not limited by the descriptionset out above but is only limited by the claims which follow, that scopeincluding all equivalents of the subject matter of the claims. Each andevery claim is incorporated into the specification as an embodiment ofthe present invention. Thus, the claims are a further description andare an addition to the preferred embodiments of the present invention.The discussion of a reference in the Background is not an admission thatit is prior art to the present invention, especially any reference thatmay have a publication date after the priority date of this application.The disclosures of all patents, patent applications, and publicationscited herein are hereby incorporated by reference, to the extent thatthey provide exemplary, procedural or other details supplementary tothose set forth herein.

For the purpose of any U.S. national stage filing from this application,all publications and patents mentioned in this disclosure areincorporated herein by reference in their entireties, for the purpose ofdescribing and disclosing the constructs and methodologies described inthose publications, which might be used in connection with the methodsof this disclosure. Any publications and patents discussed above andthroughout the text are provided solely for their disclosure prior tothe filing date of the present application. Nothing herein is to beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

Unless indicated otherwise, when a range of any type is disclosed orclaimed it is intended to disclose or claim individually each possiblenumber that such a range could reasonably encompass, including anysub-ranges encompassed therein. When describing a range of measurementsevery possible number that such a range could reasonably encompass can,for example, refer to values within the range with one significant digitmore than is present in the end points of a range. Moreover, when arange of values is disclosed or claimed, which Applicants intent toreflect individually each possible number that such a range couldreasonably encompass, Applicants also intend for the disclosure of arange to reflect, and be interchangeable with, disclosing any and allsub-ranges and combinations of sub-ranges encompassed therein.Accordingly, Applicants reserve the right to proviso out or exclude anyindividual members of any such group, including any sub-ranges orcombinations of sub-ranges within the group, if for any reasonApplicants choose to claim less than the full measure of the disclosure.

What is claimed is:
 1. A method comprising: obtaining whole blood from asubject having a level of disease mediators (y); contacting the wholeblood with an adsorbent material comprising a synthetic carbon particle(SCP) to produce a first filtrate; contacting the first filtrate with anadsorbent material comprising the SCP and an anion exchange resin wherethe weight ratio of SCP to anion exchange resin is from about 1:100 toproduce a second filtrate; contacting the second filtrate with anadsorbent material comprising the SCP and a cation exchange resin wherethe weight ratio of SCP to cation exchange resin is from about 1:100 toproduce a third filtrate; and administering the third filtrate to thesubject.
 2. The method of claim 1 wherein the subject is diagnosed withor suspected of having viruses associated with immunosuppressive events.3. The method of claim 2 wherein the viruses associated withimmunosuppressive events is a member of a viral family selected from thegroup consisting of Arenaviridae, Bunyaviridae, Filoviridae,Flaviviridae, Coronavirinae, Bunyaviridae, and Orthomyxoviridae.
 4. Themethod of claim 2 wherein the virus comprises Hantavirus,MERS-coronavirus (MERS-CoV), Influenza A virus subtype H5N1, Influenza A(H1N1) virus, Ebola Virus, Marburg virus, or combinations thereof. 5.The method of claim 1 further comprising sanitizing the SCP, the anionexchange resin, and the cation exchange resin prior to contacting withbodily fluids.
 6. The method of claim 1 further comprising contactingthe SCP, the anion exchange resin, and the cation exchange resin with acompatibilizer prior to contacting the SCP, the anion exchange resin,and the cation exchange resin with bodily fluids.
 7. The method of claim6 wherein the compatibilizer comprises a polysaccharide, a glucan,albumin, mannitol, a starch, or combinations thereof.
 8. The method ofclaim 6 wherein the compatibilizer comprises dextran.
 9. The method ofclaim 8 wherein the dextran has an average molecular weight of fromabout 1 kDa to about 500 kDa.
 10. The method of claim 6 wherein thecompatibilizer comprises hydroxyethyl starch.
 11. The method of claim 6wherein the compatibilizer comprises albumin and mannitol.
 12. Themethod of claim 1 wherein a level of a disease mediator in the thirdfiltrate is reduced by about 100% when compared to the level of diseasemediators (y).
 13. The method of claim 12 wherein the disease mediatorsare selected from the group consisting of IL-18, IFN-γ, TNF-α, IL-1β,IL-6, IL-10, MCP-1, MCSF, MIP-1 α, NO, C3a, C5a, histamine, andcombinations thereof.